Microparticle Formulations for Delivery to the Lower and Central Respiratory Tract and Methods of Manufacture

ABSTRACT

Microparticle formulations of a sialidase fusion protein are produced by contacting an aqueous solution of a protein or other active agent with an organic solvent, a counterion and a scavenging agent, and chilling the solution. The microparticles are useful for preparing stable, uniform pharmaceuticals of predetermined defined dimensions.

RELATED APPLICATIONS

This application is a continuation and claims priority to U.S.application Ser. No. 15/156,268, filed May 16, 2016, which is acontinuation and claims priority to U.S. application Ser. No.13/931,419, filed Jun. 28, 2013, which claims priority to U.S.Provisional Application Ser. No. 61/665,807, filed Jun. 28, 2012 andU.S. Provisional Application Ser. No. 61/779,653, filed Mar. 13, 2013.

BACKGROUND

The preparation and delivery of active agents including small moleculesand biologics (e.g., proteins, carbohydrates, nucleic acids, hormones,lipids) in powder or microparticle form is an area of concentratedresearch and development activity in a variety of applications includingpharmaceuticals (where the active agent is an Active PharmaceuticalIngredient (API)), nutraceuticals and cosmetics. In many cases it isdesirable for the microparticles to have a predetermined, relativelyuniform size because size can impact the deposition of themicroparticles and release of the API.

In the case of inhalable microparticle formulations it is particularlyimportant that microparticle size be controlled so that the API can bedelivered to the appropriate region(s) of the respiratory tract.Examples of diseases that can be treated by delivery of a pharmaceuticalformulation to the upper and/or central respiratory tract includerespiratory tract infections (RTIs) such as influenza, parainfluenza,RSV, sinusitis, otitis, laryngitis, bronchitis and pneumonia. Inaddition, there are large numbers of respiratory tract disorders (RTDs)that may not be caused by an infectious pathogen but affect the upperand central respiratory tract; such disorders, which can have a geneticbasis, arise due to immunodeficiencies or other (e.g., α-1-antitrypsin)deficiencies, result from exposure to allergens and/or chemicalpollutants, or present as complications of infectious diseases such asthe RTIs described above or inflammatory diseases such as inflammatorybowel syndrome and Crohn's disease include allergic and non-allergicasthma, COPD, bronchiectasis, vasculitis, mucous plugging, Wegener'sgranulomatosis and cystic fibrosis (CF).

In some cases, for example, when an infection is present in the lowerrespiratory tract, it can be desirable to administer a formulationcontaining microparticles that are delivered to the lower and centralrespiratory tract. Thus, in some cases it can be desirable to administera formulation containing microparticles that have an MMAD of 3-8 micronsor 5-7 microns. The smaller MMAD of the microparticles in suchformulations can increase the fraction of microparticles that are smallenough to become absorbed into the bloodstream. Since systemic exposureis sometimes not desirable, the formulations with a relatively smallMMAD can pose increase risk of unwanted systemic exposure. However,tight control over the range of the particle size can reduce the risk.Moreover, for some patients, e.g., immunocompromised patients, sufferingfrom influenza or parainfluenza, the importance of delivering therapy tothe lower respiratory tract justifies a potential increase in risk of asmall degree of systemic exposure.

For the treatment of RTIs and RTDs, the microparticles of the drug mustbe small enough to be deposited and act at the desired target site inthe upper and/or central respiratory airways of the lungs (e.g., theepithelial cells of the upper respiratory tract in the case ofinfluenza; the central respiratory tract in the case of asthma or COPD),yet not be so small as to reach deeper parts of the lungs, such as thealveoli, become absorbed into the bloodstream, and compromise thepharmacokinetic profile or even the safety of the drug. Accordingly,there is a need for a method of producing microparticle formulationswhose size can be fine-tuned for delivery to a target site of interestwhile reducing delivery to sites that are undesirable.

There further is a need for microparticle formulations in which theincorporated active agent is stable for relatively long periods of time.For stability, the active agent in the formulation should not react withor otherwise be degraded by other ingredients/excipients in theformulation (for example, compounds that improve bioavailability,delivery, safety, etc. of the active agent). The active agent in theformulation should also be protected from components that may be presentin the packaging materials and/or delivery systems, e.g., medicaldevices, containers, capsules or gels. For example, when the activeagent is a protein, aldehydes and other cross-linking agents that may bepresent in some packaging materials or arise as byproducts during themanufacture of the materials can react with the active agent to formprotein aggregates or oligomers. There is a need for microparticleformulations that can protect the active agent against components ofpackaging materials that could compromise its stability.

SUMMARY

Provided herein are methods of producing relatively uniform sizedmicroparticle formulations of a macromolecule or small molecule activeagent. In some cases, the resulting microparticles are of apredetermined size and have a narrow range of size distribution(geometric standard deviation (“GSD”) between 1.2-2.0, e.g., 1.5-1.7).The methods provided herein include the steps of mixing together asolution of an active agent in an aqueous solvent, a counterion and asolvent (e.g., isopropanol) and cooling the resulting mixture (alsoreferred to herein as cocktail solution or feedstock solution) to apredetermined temperature below about 25° C. at a cooling rate that ismaintained at a constant fixed value until the mixture is at apredetermined temperature below about 25° C. In many cases there are twoor more cooling phases during which the temperate is decreased at afixed rate. In some cases the solution is held for a period of time at apredetermined temperature. The resulting microparticles can be separatedfrom the mixture to remove components other than the microparticles by,for example, sedimentation, filtration and/or freeze-drying.

The size of the microparticles of the resulting formulations iscontrolled, in large part, by a combination of the choice of counterionand the cooling rate. In general, the faster the cooling rate, thesmaller the size of the resulting microparticles. The uniformity of thesize is achieved in certain cases by maintaining the cooling rate at aconstant, fixed value until the mixture is cooled to the desiredpredetermined temperature below about 25° C. Thus, the cooling rate ismaintained regardless of the changing temperature differential duringcooling, i.e., the difference between the temperature of the cocktailsolution at any given time during the cooling process and the finalpredetermined temperature to which it is cooled.

In general, in the case of the sialidase fusion protein DAS181, whosesequence is set forth in SEQ ID NO:1 (no amino terminal methoionine) andSEQ ID NO:2 (amino terminal methionine present), when the methods employfaster cooling rates and counterions such as sodium sulfate or magnesiumsulfate, smaller microparticles (value between about 3 microns to about8 microns mass median aerodynamic diameter (MMAD)) of DAS181 are formed.

In particular embodiments, provided herein are methods of making uniformmicroparticle formulations of DAS181 with a mass median aerodynamicdiameter (MMAD) that has a value between about 8.0 microns to about 3.0microns (preferably 7.5-4.5 microns) and a geometric standard deviation(GSD) of between about 1.2 to about 2.0 (e.g., 1.5-1.7). As discussedfurther below, in some cases it is desirable that microparticles ofDAS181 or other active agents for delivering microparticles to thecentral and lower respiratory tract for treating respiratory tractinfections or disorders such as influenza, parainfluenza, asthma or COPDhave a size, generally 4 microns to 10 microns, which permits theirdeposition at locations that are the targets of the disease, such as thethroat, trachea and bronchi, while reducing deposition in the deep lung,e.g., alveoli and/or absorption into the blood stream. In many cases,the site of deposition depends on the inhaler flow rate. In someembodiments the particles are inhaled at 50-701/min (e.g., 55-65 l/minor 60 l/min).

In some embodiments, the methods provided herein include citric acid orcitrate as the counterion and a constant fixed cooling rate of betweenabout 0.3° C./min to about 0.8° C./min to cool the feedstock solutionfrom a temperature of about 25° C. to a predetermined temperature ofabout −45° C., −50° C., −55° C., or −60° C., whereby a microparticleformulation of DAS181 that has a particle size (MMAD) of about 6.0, 6.1,6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 or 7.0 microns and a GSD of about1.2-2.0 is formed.

In one embodiment, a uniform formulation of DAS181 microparticles ofmass median aerodynamic diameter (MMAD) about 6.0, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9 or 7.0 microns and a GSD of 1.5-1.7 is preparedby cooling a cocktail solution comprising DAS181 as the active agent,magnesium sulfate as a counterion and isopropanol as the organic solventfrom a temperature of about 25° C. to a temperature of −45° C. at acooling rate of −0.5° C./min.

Also provided are microparticle formulations prepared according to themethods provided herein. In some embodiments, the active agent or API inthe formulation is for treating diseases that affect the respiratorytract such as influenza, asthma and COPD.

An example of an API that can be used to treat respiratory diseases anddisorders is the sialidase fusion protein, DAS181. The DAS181 fusionprotein can be expressed by various expression systems. As a result, itcan be present and functional in the two forms: one in which thepolypeptide begins with a methionine (SEQ ID NO:2) or one in which thepolypeptide lacks the initial methionine and begins with valine (SEQ IDNO:1). Hence, in an example where the API is DAS181, the API componentcan be the polypeptide of SEQ ID NO:1 or the polypeptide of SEQ ID NO:2or it can be a combination comprising the polypeptide of SEQ ID NO:1 andthe polypeptide of SEQ ID NO:2.

In general, DAS181 can be: A) a polypeptide comprising (or consisting ofor consisting essentially of) the amino acid sequence of SEQ ID NO:1; B)a polypeptide comprising (or consisting of or consisting essentially of)the amino acid sequence of SEQ ID NO:2; or C) a mixture of apolypeptides comprising (or consisting of or consisting essentially of)SEQ ID NO:1 and polypeptides comprising (or consisting of or consistingessentially of) SEQ ID NO:2.

For administering DAS181 to the central respiratory tract (asthma, COPD)or to the upper respiratory tract (influenza, parainfluenza), it isdesirable to have uniform microparticle formulations with particles ofmass median aerodynamic diameter (MMAD) between about 3 microns to about8 microns and a GSD of between about 1.2 to 2.0. In general, dependingsomewhat on the flow rate of the inhalation, particles with MMAD betweenabout 3 microns to about 5 microns are expected to deposit in the lowerrespiratory tract, particles with MMAD between about 5 microns to about8 microns are expected to deposit in the central to upper respiratorytract, and particles of about 8 microns to about 10 or 11 microns areexpected to deposit primarily in the upper respiratory tract.

Microparticles that are smaller than about 2 or 2.5 microns can reachalveoli of the lungs and release the drug, where it can be absorbed intothe bloodstream. In some cases it is desirable that particles smallerthan about 2.0 or 2.5 microns are present at a very low level or areessentially absent from pharmaceutical formulations not intended forpulmonary delivery or systemic absorption.

Thus, in some embodiments, provided herein are uniform-sizedmicroparticle formulations of DAS181 (polypeptides comprising orconsisting of SEQ ID NO:1, polypeptides comprising or consisting of SEQID NO:2 or in some instances a combination comprising polypeptidescomprising or consisting of SEQ ID NO:1 and polypeptides comprising orconsisting of SEQ ID NO:2) or other active agents useful for preventingor treating infections and other disorders of the respiratory tract suchas influenza, parainfluenza, asthma and COPD, wherein the microparticlesare of a size that is suitable for deposition in the central to lowerrespiratory tract. For optimal deposition of the microparticles attarget sites of the infection or disorder (upper respiratory forinfluenza, central respiratory for asthma), the majority of themicroparticles in the formulation must not be (a) so big that they aretrapped at the front end in the mouth (e.g., greater than about 10.5 or11 microns); or (b) so small that they are deposited deep in the lungsand absorbed systemically into the blood stream through the alveoli(e.g., less than about 2.0 or 2.5 microns).

In many cases, the microparticles themselves are relative homogeneous,i.e., the formulation components, e.g., the API (DAS181) are notsegregated and are instead generally evenly distributed throughout themicroparticles. Further, the fine particle fraction (FPF) containingmicroparticles that are smaller than the desired size for deposition tothe target site of interest is less than 10%, generally less than about8%, 7%, 6%, 5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5% or 1%.

In many cases, the microparticles themselves are essentially homogenouswith respect to the API, i.e., the formulation components, e.g., the APIis essentially one specific polypeptide of DAS181 of one sequence, e.g.,SEQ ID NO: 1 or SEQ ID NO: 2. In such homogenous batches, the API is notsegregated and is instead generally evenly distributed throughout themicroparticles.

In certain cases, the microparticles themselves are heterogeneous withrespect to the API, i.e., the formulation components, e.g., the API iscomprised of one or more than one ingredient, i.e., polypeptides ofDAS181 of two different sequences, e.g., SEQ ID NO: 1 together in abatch SEQ ID NO: 2. In such heterogeneous batches, the heterogeneous APIpolypeptides are not segregated and are instead generally evenlydistributed throughout the microparticles.

Microparticle Formulation II

In some embodiments, DAS181 (comprising or consisting of SEQ ID NO:1 orcomprising or consisting of SEQ ID NO:2 or in some instances, a batchwith polypeptides comprising or consisting of SEQ ID NO:1 andpolypeptides comprising or consisting of SEQ ID NO:2) is provided in thefollowing microparticle formulation:

Formulation II—not anhydrous (wt/wt %):

-   -   a) DAS181: 64.5-64.7% of SEQ ID NO:1 or SEQ ID NO:2 or a batch        comprising SEQ ID NO:1 and SEQ ID NO:2    -   b) Histidine free base: 4.3-4.6%    -   c) Histidine HCl: 5.8-6.3%    -   d) Trehalose: 9.0-9.7%    -   e) Magnesium sulfate: 4.6-5.9%    -   g) Water: 10.0% (depending on humidity of storage conditions)        The microparticles can also include small amounts of sodium        acetate (less than 1%, less than 0.5%, less than 0.1%, less than        0.05%, e.g., 0.03%); small amounts of calcium chloride (less        than 1%, less than 0.5%, less than 0.3%, e.g., 0.3%); and small        amounts of acetic acid (less than 1%, less than 0.5%, less than        0.1%, less than 0.05%, e.g., 0.02%). Small amounts of residual        isopropanol can sometimes be present (less than 1%, less than        0.5%, less than 0.1%, less than 0.05%, less than 0.01%).

Formulation II—anhydrous (wt/wt %):

-   -   a) DAS181: 71.7-71.9% of SEQ ID NO:1 or SEQ ID NO:2 or a batch        comprising SEQ ID NO:1 SEQ ID NO:2    -   b) Histidine free base: 4.8-5.1%    -   c) Histidine HCl: 6.5-7.0%    -   d) Trehalose: 10.7-10.1%    -   e) Magnesium sulfate: 5.1-6.5%        The microparticles can also include small amounts of sodium        acetate (less than 1%, less than 0.5%, less than 0.1%, less than        0.05%, e.g., 0.03%); small amounts of calcium chloride (less        than 1%, less than 0.5%, less than 0.3%, e.g., 0.3%); and small        amounts of acetic acid (less than 1%, less than 0.5%, less than        0.1%, less than 0.05%, e.g., 0.02%). Small amounts of residual        isopropanol can sometimes be present (less than 1%, less than        0.5%, less than 0.1%, less than 0.05%, less than 0.01%). In some        embodiments the formulation is free of citrate.

In one embodiment the method of preparing Formulation II includes thefollowing steps:

(a) An Excipient Solution (pH 6) containing histidine, trehalose,magnesium sulfate and calcium chloride is prepared by combining stocksolutions is prepared and sterile filtered.

(b) The Excipient Solution is added, with mixing, to a compoundingvessel containing DAS181 protein (SEQ ID NO:1 or SEQ ID NO:2 or a batchcomprising SEQ ID NO:1 and SEQ ID NO:2) at 125 mg/ml initialconcentration of DAS 181.

(c) Sterile filtered isopropanol is added to the compound vessel withmixing to form the Feedstock Solution. The final composition of theFeedstock Composition is as follows: 70 mg/ml DAS181 (SEQ ID NO:1 or SEQID NO:2 or a batch with polypeptides comprising SEQ ID NO:1 andpolypeptides comprising SEQ ID NO:2), 25% isopropanol, 4.99 mg/mlhistidine, 6.80 mg/ml histidine-HCl, 10.50 mg/ml trehalose, 5.06 mg/mlmagnesium sulfate, 0.21 mg calcium chloride, 0.05 mg/ml sodium acetate,0.02 mg/ml acetic acid. The pH of the solution is 6.0. The time betweeninitiating the addition of isopropanol and starting the lyophilizationcycle is between 60 minutes.

(e) Stainless Steel trays that have undergone depyrogenation are eachfilled with 18 g of the Feedstock Solution, using a metering pump.

(f) The filled Stainless Steel trays are subjected to a LyophilizationCycle as follows:

-   -   a. the trays are gasketed and placed in the lyophilizer shelves        at 25° C. for 5 minutes;    -   b. the temperature of the shelves is lowered to −45° C. at a        rate of −0.5° C./minute;    -   c. primary drying is accomplished by setting the condenser to        less than −80° C., applying a vacuum of 125 mTorr and increasing        the temperature to −0° C. at a ramp rate of 1° C./minute and        then holding for 60 hrs;    -   d. the secondary drying is accomplished increasing the        temperature to 30° C. at a rate of 1° C./minute and then holding        for 6 hrs; and    -   e. the vacuum is released and the lyophilizer is backfilled with        nitrogen to prevent oxidation of the microparticle formulation        before transferring into bottles for bulk mixing and aliquoting        the bulk powder for storage at <−15° C.

Microparticles of Formulation II can have one or more of: an MMAD of 6.5microns (or 2-8 microns. 3-8 microns or 5-7), a GSD of 1.5-1.7 (or1.3-1.9 or 1.4-1.8), a FPF (volume % below 5 microns) of 6.6% (less than35%, 30%, 25%, 20%, 15%, 10% or 5%) and, a Tg of 38° C.

Thus, in some embodiments, the microparticles have a MMAD of 3-8 (5-7)microns (6.2-6.8 microns) with a GSD of 1.3-1-6 (1.4-1.6), a FPF of lessthan 9% (less than 8%, less than 7%, about 6-7%) and comprise (on aweight % basis) DAS181 (SEQ ID NO:1 or SEQ ID NO:2 or a batch comprisingSEQ ID NO:1 and SEQ ID NO:2): 60-70% (62-68%, 64-66%, 65%); Histidinefree base: 3-6% (4-5%); Histidine HCl: −5-9% (5-7%, 8-9%); Trehalose:7-11% (8-10%, 8.5-9.5%); Magnesium sulfate: 4-8% (5-7%, 4-6%); andWater: 6-12% (8-12%, 9-11%). The microparticles can also include smallamounts of sodium acetate (less than 1%, less than 0.5%, less than 0.1%,less than 0.05%, e.g., 0.03%); of calcium chloride (less than 1%, lessthan 0.5%, less than 0.4%, less than 0.3%, e.g., 0.1-0.3%) and smallamounts of acetic acid (less than 1%, less than 0.5%, less than 0.1%,less than 0.05%, e.g., 0.01%). Small amounts of residual isopropanol cansometimes be present (less than 1%, less than 0.5%, less than 0.1%, lessthan 0.05%, less than 0.01%).

When the microparticles of Formulation II are anhydrous they cancomprise (on a weight % basis) DAS181 (SEQ ID NO:1 or SEQ ID NO:2 or abatch comprising SEQ ID NO:1 and SEQ ID NO:2): 69-74% (70-73%, 71-72%,72%); Histidine free base: 3-8% (4-7%, 4-6%); Histidine HCl: 4-9% (5-8%,6-7%); Trehalose: 8-12% (9-11%, 8-10%); Magnesium sulfate: 4-8% (5-7%,6-7%). The microparticles can also include small amounts of sodiumacetate (less than 1%, less than 0.5%, less than 0.1%, less than 0.05%,e.g., 0.03%); small amounts of calcium chloride (less than 1%, less than0.5%, less than 0.1%, less than 0.05%, e.g., 0.03%) and small amounts ofacetic acid (less than 1%, less than 0.5%, less than 0.1%, less than0.05%, e.g., 0.01%). Small amounts of residual isopropanol can sometimesbe present (less than 1%, less than 0.5%, less than 0.1%, less than0.05%, less than 0.01%).

Microparticle Formulation I

In some embodiments, DAS181 (comprising or consisting of SEQ ID NO:1 orcomprising or consisting of SEQ ID NO:2 or in some instances, a batchwith polypeptides comprising or consisting of SEQ ID NO:1 andpolypeptides comprising or consisting of SEQ ID NO:2) is provided in thefollowing microparticle formulation:

Formulation I—not anhydrous (wt/wt %):

-   -   a) DAS181: 86.7% of SEQ ID NO:1 or SEQ ID NO:2 or a batch        comprising SEQ ID NO:1 and SEQ ID NO:2)    -   b) sodium sulfate 2.5%    -   c) Water:10.0% (depending on humidity of storage conditions)        The microparticles can also include small amounts of sodium        acetate (less than 1%, less than 0.5%, less than 0.1%, less than        0.05%, e.g., 0.03%); small amounts of calcium chloride (less        than 1%, less than 0.5%, less than 0.1%, less than 0.05%, e.g.,        0.03%) and small amounts of acetic acid (less than 1%, less than        0.5%, less than 0.1%, less than 0.05%, e.g., 0.01%). Small        amounts of residual isopropanol can sometimes be present (less        than 1%, less than 0.5%, less than 0.1%, less than 0.05%, less        than 0.01%).

Formulation I—anhydrous (wt/wt %):

-   -   a) DAS181: 96.3% SEQ ID NO:1 or SEQ ID NO:2 or a batch        comprising SEQ ID NO:1 and SEQ ID NO:2)    -   b) sodium sulfate 2.7%        The microparticles of Formulation I can also include small        amounts of sodium acetate (less than 1%, less than 0.8%, less        than 0.7%, less than 0.6%); small amounts of calcium chloride        (less than 1%, less than 0.5%, less than 0.4%) and small amounts        of acetic acid (less than 1%, less than 0.5%, less than 0.4%,        less than 0.3%, e.g., 0.2%). Small amounts of residual        isopropanol can sometimes be present (less than 1%, less than        0.5%, less than 0.1%, less than 0.05%, less than 0.01%).

Microparticles of Formulation I can have one or more of: an MMAD of 4-7microns (or 2-8 microns. 3-8 microns or 5-7 microns), a GSD of 1.5-1.7(or 1.3-1.9 or 1.4-1.8), a FPF (volume % below 5 microns) of 8% (lessthan 35%, 30%, 25%, 20%, 15%, 10% or 5%) and, a Tg of 38° C.

Thus, in some embodiments, the microparticles have a MMAD of 3-8 (5-7)microns (6.2-6.8 microns) with a GSD of 1.3-1-6 (1.4-1.6), a FPF of lessthan 9% (less than 8%, less than 7%, about 6-7%) and comprise (on aweight % basis) DAS181 (SEQ ID NO:1 or SEQ ID NO:2 or a batch comprisingSEQ ID NO:1 and SEQ ID NO:2):

Microparticle Formulations

The microparticle formulations obtained by the methods provided hereinare of a relatively uniform size distribution, i.e., relativelymonodisperse, with a geometric standard deviation (GSD) of between about1.2 and 2.0, generally between about 1.2 and 1.5, 1.6, 1.7 or 1.8. Theparticles are also homogeneous, i.e., the formulation components are notsegregated and are evenly distributed throughout the particles. Further,the fine particle fraction (FPF) containing microparticles that aresmaller than 5 microns is less than 10%, generally less than about 8%,7%, 6%, 5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5% or 1%.

In some embodiments, in addition to DAS 181 SEQ ID NO:1 or SEQ ID NO:2or a batch comprising SEQ ID NO:1 and SEQ ID NO:2), microparticles caninclude an additional active agent which can be a macromolecule such asa protein, a nucleic acid, a carbohydrate, a lipid, a fatty acid, apolysaccharide or a carbohydrate- or polysaccharide-protein conjugate.In other embodiments, the active agent or API can be a small moleculesuch as a prostaglandin, an antibiotic selected from amongaminoglycosides, ansamycins, carbacephem, carbapenems, cephalosporins,macrolides, penicillins, quinolones, sulfonamides and tetracyclines, anantiviral agent such as zanamivir or oseltamivir phosphate, or achemotherapeutic agent selected from among alkylating agents,anthracyclines, cytoskeletal disruptors, epothilones, inhibitors oftopoisomerase II, nucleotide analogs, platinum-based agents, retinoidsand vinca alkaloids.

In the methods provided herein, the microparticle formulation of theprotein can also be tailored to a desired predetermined particle sizeand uniform size distribution by modifying one or more additionalparameters or steps, including one or more of the following: (1) theconcentration of the protein in the feedstock solution; (2) the natureand/or concentration of the counterion in the feedstock solution; (3)the nature and/or concentration of the organic solvent in the feedstocksolution; (4) the concentration of excipient; (5) the volume offeedstock solution in the lyophilization trays; and (6) one or morecontrolled temperature ramping (temperature decreasing and/ortemperature increasing) steps for forming and isolating the resultingmicroparticles.

Also provided herein are methods of making stable microparticleformulations and the resulting stable microparticle formulations inwhich the active agent (or API, for a pharmaceutical formulation) isprotected from degradation or aggregation resulting from materialspresent in the packaging container or delivery system. The methodsprovided herein include the steps of: mixing together: (i) a solution ofan active agent in an aqueous solvent, (ii) a counterion selected fromamong citric acid/citrate, magnesium sulfate, potassium sulfate orcalcium sulfate, phosphate, pivalate, rubidium, bromine, perchlorate,itaconate, and any salt, acid, or base form thereof, (iii) one or morescavenging agents and (iv) an organic solvent (e.g., isopropanol); andcooling the resulting mixture (also referred to herein as cocktailsolution or feedstock solution) to a predetermined temperature belowabout 25° C. either gradually or at a cooling rate that can bemaintained at a constant fixed value until the mixture is at thepredetermined temperature below about 25° C., whereby a compositioncontaining microparticles that include DAS181 SEQ ID NO:1 or SEQ ID NO:2or a batch comprising SEQ ID NO:1 and SEQ ID NO:2) at about 50% to about85% wt/wt, the counterion at about 2% to about 6% wt/wt and thescavenging agents at about 8% to about 40% wt/wt is formed. Theresulting microparticles can be separated from the mixture to removecomponents other than the microparticles by, for example, sedimentation,filtration and/or freeze-drying.

The resulting dry microparticles, in certain embodiments, can have acomposition of between about 50% to about 75% wt/wt of SEQ ID NO:1 orSEQ ID NO:2 or a batch comprising SEQ ID NO:1 and SEQ ID NO:2), betweenabout 2% to about 6% wt/wt of the counterion, between about 5% to about40% wt/wt of each scavenging agent and between about 5% to about 10%residual moisture. The scavenging agent can be a primary or secondaryamine, a chelator, an antioxidant, a sugar, or combinations thereof andgenerally is present in at least a 100-fold excess, upto about a 150,200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850,900, 950 or a 1000 fold or more molar excess relative to the aldehyde(s)present in the material used to package the microparticles. Thescavenging agent is incorporated into the microparticles obtained by themethods provided herein, and can protect the active agent against thedamaging effects of some materials, such as aldehydes, that may bepresent in a packaging container or delivery system such as HPMCcapsules, certain gel capsules, pullulan polysaccharide capsules andaluminum foil laminate blister packs.

In some embodiments of the method, the active agent is a protein and inparticular embodiments, the protein is DAS181 SEQ ID NO:1 or SEQ ID NO:2or a batch comprising SEQ ID NO:1 and SEQ ID NO:2). In otherembodiments, the scavenging agent is histidine. In yet otherembodiments, the scavenging agent is tryptophan. In further embodiments,combinations of scavenging agents, such as an amino acid and a sugar,more than one amino acid, or more than one amino acid and a sugar, areused; in particular embodiments, the combinations of scavenging agentsare histidine and trehalose, histidine and sucrose, glycine and sucrose,histidine, glycine and sucrose, or histidine and tryptophan. In someembodiments, the microparticle formulations containing an active agent,a counterion and a scavenging agent as provided herein can further betailored to a desired predetermined particle size and uniform sizedistribution by applying a constant, fixed cooling rate to the feedstocksolution for cooling the feedstock solution from a temperature above orat 25° C. to a predetermined temperature below 25° C., wherebymicroparticles are formed. Additional parameters or steps that can beused to obtain a desired particle size and/or size distribution caninclude one or more of the following: (1) the concentration of theactive agent in the feedstock solution; (2) the nature and/orconcentration of the counterion in the feedstock solution; (3) thenature and/or concentration of the organic solvent in the feedstocksolution; (4) the concentration of excipient; (5) the volume offeedstock solution in the lyophilization trays; and (6) one or morecontrolled temperature ramping (temperature decreasing and/ortemperature increasing) steps for forming and isolating the resultingmicroparticles.

Also provided herein are microparticles of a protein containing betweenabout 50% to about 85% wt/wt of the protein, about 2% to about 6% wt/wtof a counterion selected from among citric acid/citrate, magnesiumsulfate, potassium sulfate or calcium sulfate, phosphate, pivalate,rubidium, bromine, perchlorate, itaconate, and any salt, acid, or baseform thereof, and about 8% to about 40% wt/wt of one or more scavengers.In some embodiments, the protein is DAS181 (SEQ ID NO:1 or SEQ ID NO:2or a batch with polypeptides comprising SEQ ID NO:1 and polypeptidescomprising SEQ ID NO:2) and in further embodiments, the scavenging agentis present in about 9% to about 11% wt/wt of the dry powdermicroparticles. In some embodiments, the scavenging agent(s) present inthe DAS181 microparticle formulations is histidine, histidine/histidineHCl, histidine/trehalose, histidine/tryptophan, histidine/sucrose,glycine/sucrose, or histidine/glycine/sucrose.

Exemplary feedstock solutions used to prepare microparticle formulationsof DAS181 according to the methods provided herein can include betweenabout 10 mg/ml to about 70 mg/ml DAS181 (SEQ ID NO:1 or SEQ ID NO:2 or abatch with polypeptides comprising SEQ ID NO:1 and polypeptidescomprising SEQ ID NO:2) and one or more of the following: histidine fromabout 0.1-about 11 mg/ml, glycine from about 0.006-about 7 mg/ml,trehalose or sucrose from about 1.93-about 11 mg/ml, tryptophan fromabout 1.11-about 3.5 mg/ml, magnesium sulfate from about 0.24-about 6.8mg/ml, calcium chloride from about 0.03-about 0.21 mg/ml.

Also provided herein are uniform, stable microparticle formulations ofDAS181 that include a counterion at 2% to about 6% wt/wt, a scavengingagent that is an amine at about 9% to about 20% wt/wt, and a sugar. Alsoprovided herein are microparticle formulations containing an activeagent, a counterion selected from among citric acid/citrate, magnesiumsulfate, potassium sulfate or calcium sulfate, phosphate, pivalate,rubidium, bromine, perchlorate, itaconate, and any salt, acid, or baseform thereof and a scavenging agent that is a chelator or anantioxidant. In some embodiments, the scavenging agent is ascorbic acidor ascorbic acid phosphate; in other embodiments, the scavenging agentis TETA. In further embodiments, the active agent is a protein.

The methods provided herein for making microparticle formulations caninclude additional steps. For example, after cooling the feedstocksolution at a constant, fixed rate to a predetermined temperature belowabout 25° C. for forming the microparticles, the cooled solution can beheld at that predetermined temperature for a specified period of time toremove unincorporated components of the microparticles by freeze-drying.The microparticles can further be heated in primary and optionallysecondary drying steps to remove volatile components by sublimation. Insome embodiments, the resulting microparticles are further packaged intomaterials containing formaldehyde and/or other aldehydes, such ashydroxypropyl methylcellulose (HPMC), aluminum foil laminates, gelcapsules or pullulan polysaccharide.

Also provided herein are microparticle formulations that are packaged inmaterials containing formaldehyde and/or other aldehydes. Such materialsinclude, but are not limited to, hydroxypropyl methylcellulose (HPMC)capsules, certain gel capsules, aluminum foil laminate blister packs andpullulan polysaccharide capsules.

Also provided herein are articles of manufacture that contain amicroparticle formulation that includes a sialidase or a sialidasefusion protein in an amount of about 60% to about 75% wt/wt, acounterion and a scavenging agent that is a primary amine in the amountof about 8% to about 11% wt/wt, a packaging material for theformulation, where the material contains formaldehyde and/or otheraldehydes, and a label that indicates that the composition is for atherapeutic indication. In one embodiment, the therapeutic indication isinfluenza. In other embodiments, the therapeutic indication is asthma orCOPD. In yet other embodiments, the therapeutic indication is selectedfrom among parainfluenza, RSV, sinusitis, otitis, laryngitis,bronchitis, pneumonia, bronchiectasis, vasculitis, mucous plugging,Wegener's granulomatosis and cystic fibrosis (CF). In some embodiments,the scavenging agent is histidine; in other embodiments, the scavengingagent is a combination of histidine and trehalose. In yet otherembodiments, the counterion is selected from among citric acid/citrate,magnesium sulfate, potassium sulfate or calcium sulfate, phosphate,pivalate, rubidium, bromine, perchlorate, itaconate, and any salt, acid,or base form thereof. The packaging material can be a HPMC capsule; infurther embodiments, the HPMC capsule is clear. In other embodiments,the packaging material can be a gel capsule, or a pullulanpolysaccharide capsule. In yet other embodiments, the article ofmanufacture further contains a secondary packaging material; in someembodiments, the secondary packaging material is a foil laminate; inparticular embodiments, the foil laminate is a cold form foil aluminumlaminate blister pack. In some embodiments, the scavenging agent is anamine; in further embodiments, the amine is selected from among lysine,histidine, glycine, arginine, glutamine, glutamic acid, cysteine,alanine, tyrosine, tryptophan, aminoguanidine, cysteamine, serine,carnosine, hydralazine and poly(l-lysine). In one embodiment, thesialidase fusion protein is DAS181 having the sequence set forth in SEQID NO:1 or SEQ ID NO:2 or, in some instances, DAS181 is present as abatch with polypeptides comprising SEQ ID NO:1 and polypeptidescomprising SEQ ID NO:2; in a particular embodiment, the sialidase fusionprotein is DAS181 having the sequence set forth in SEQ ID NO:1 or SEQ IDNO:2 or, in some instances, DAS181 is present as a batch withpolypeptides comprising SEQ ID NO:1 and polypeptides comprising SEQ IDNO:2 and the scavenging agent is histidine or a combination of histidineand trehalose.

The articles of manufacture provided herein also can contain an inhalerfor pulmonary administration of the composition. In certain embodiments,the inhaler is a dry powder inhaler, a metered dose inhaler or anelectrostatic delivery device.

DETAILED DESCRIPTION A. Definitions

The term “microparticle” as used herein is interchangeable with“microsphere” and refers to particles in the size range (average length,width or diameter) of about or at 0.001 micron (μm) to about or at 500microns that contain a macromolecule or small molecule that is an activeagent of interest, such as a drug or nutritional supplement. Among themicroparticles provided herein are those of a size between about 3microns to about 8 microns mass median aerodynamic diameter (MMAD) andcontaining active agents, including proteins and, in some embodiments,the sialidase fusion protein DAS181 (SEQ ID NO:1 or SEQ ID NO:2 or abatch with polypeptides comprising SEQ ID NO:1 and polypeptidescomprising SEQ ID NO:2), which are for treating respiratory tractdiseases of the upper and/or central respiratory tract. The active agentcan be a small molecule or a macromolecule. The macromolecule, forexample, a protein, nucleic acid, lipid or polysaccharide, or themacromolecule forming the microparticle can be the active agent or canbe a carrier for the active agent, such as a drug or a nutritionalsupplement. The microparticles also can contain synthetic macromoleculesincluding polymers, such as polyethylene glycol (PEG), polylactic acid(PLA), polylactic-co-glycolic acid (PLGA), and natural polymers such asalbumin, gelatin, chitosan and dextran.

The term “microparticle” as used herein also generally refers to aparticle that is not a solid form of the entire solution from which itis produced; rather, the microparticle as used herein generally is anassembly of a fraction of the components of a solution, including activeagents, salts, counterions, solvents, scavenging agents and otheringredients and is formed by a process including, but not limited to,precipitation, sedimentation, phase separation and colloid formation.

The term “dry” or “dry powder” formulation in reference tomicroparticles, as used herein refers to microparticle formulations thatare separated from unincorporated components of the feedstock solutionby a process including, but not limited to, precipitation,freeze-drying, sedimentation, phase separation, colloid formation anddrying to sublime volatiles in the feedstock solution. The dry powderformulations of microparticles provided herein can include residualmoisture, generally at about 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5% orless

The term “mixture” is used interchangeably with “cocktail solution” or“feedstock solution” herein and refers to the homogeneous mixturedistribution of ingredients obtained just prior to lyophilization toform the microparticle formulation; the distinct ingredients of thefeedstock solution are recognizable only at the molecular level.

As used herein, “shelf life” or “stability” refers to the time afterpreparation of the microparticle composition that the compositionretains at least about or 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% of the initial activity that is present in thecomposition and other general characteristics of the microparticles suchas no more than about 1-2% aggregate formation (e.g., dimers and higherorder oligomer formation) over time and the retention of size, shape,color and aerodynamic particle size distribution. Thus, for example, acomposition that is stable for or has a shelf life of 30 days at roomtemperature, defined herein as range of between about 18° C. to about25° C., 26° C., 27° C. or 28° C., would have at least about 70%, 80%,85%, 90% 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the initialamount of the activity of protein present in the composition at 30 daysfollowing storage at 18° C. to about 25° C., 26° C., 27° C. or 28° C.The shelf life of the microparticle compositions provided hereingenerally is at least about 10 days at 55° C., at least about 2-3 weeksat 42° C., at least about 6-8 weeks at 37° C., and at least about eightmonths or greater at 25° C.; however, microparticles compositions of anylength of shelf life at any temperature that are produced by the methodsprovided herein are contemplated herein.

As used herein, “organic solvent” refers to a solvent that is an organiccompound, which is any member of a large class of chemical compoundswhose molecules contain carbon and hydrogen. Such solvents can include,for example, compounds from the following classes: aliphatic or aromaticalcohols, polyols, aldehydes, alkanes, alkenes, alkynes, amides, amines,aromatics, azo compounds, carboxylic acids, esters, dioxanes, ethers,haloalkanes, imines, imides, ketones, nitriles, phenols and thiols. Insome embodiments of the methods provided herein, the organic solventsused are not polymers. In other embodiments, the organic solvent used isa solvent other than ethanol.

As used herein, an “aqueous solvent” refers to water, or a mixture ofsolvents that contains at least about 50% or 50%, at least about 60% or60%, at least about 70% or 70%, or about or at 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99% or higher amounts of water. The term “aqueoussolvent” as used herein also refers to solutions containing water as asolvent, such as buffers, salt solutions, solutions containingcounterions, and other solutes that are soluble in water.

As used herein, the term “counterion” refers to a charged orcharge-polarizable molecule that can initiate formation of amicroparticle from a macromolecule, such as a protein, nucleic acid,lipid or oligosaccharide. For example, in the case of the DAS181 fusion(e.g., SEQ ID NO:1 or SEQ ID NO:2 or a batch comprising SEQ ID NO:1 andSEQ ID NO:2), sodium sulfate, magnesium sulfate and citric acid aresuitable counterions because they can initiate the formation ofmicroparticles in the methods provided herein. The suitability of acharged molecule as a counterion can be determined empirically based onparameters including, but not limited to, the type of protein, the pH,the ionic strength, the type of organic solvent used, and the presenceof salts and additional ingredients including the active agent(s). Asprovided and described herein, counterions can be anionic or having anet negative charge or charge-polarizable group(s), cationic or having anet positive charge or charge-polarizable group(s), or zwitterionic andpossessing both negative and positive charged or charge-polarizablegroups.

As used herein, the term “cooling” at a “constant, fixed (or preset)rate” means that the cooling rate is set at a predetermined value (i.e.,fixed, or preset) and this rate is then applied, within a reasonablevariation of about +/−10-15% of the preset value, throughout the coolingprocess (i.e., constant). Thus, when the feedstock solution from whichthe microparticles are formed is cooled from a temperature above or atabout 25° C. to a predetermined temperature below about 25° C. to formthe microparticles, the cooling rate is maintained at the same valueregardless of the changing temperature differential during cooling,i.e., regardless of the difference between the temperature of thecocktail solution at any given time during the cooling process and thefinal predetermined temperature to which it is cooled. The fixed,constant cooling rate at which the feedstock solution is cooled to apredetermined temperature below about 25° C. can be preset by a computerprogram (e.g., programming the lyophilizer to cool the feedstocksolution at a preset, fixed rate) or by mechanical means, e.g., stirringthe feedstock solution in a manner that maintains a constant coolingrate. The value of the fixed cooling rate can be selected depending onthe desired size of the microparticles and generally can be betweenabout 0.1° C./min to about 1° C./min. For example, for microparticleformation of a protein, such as DAS181 (SEQ ID NO:1 or SEQ ID NO:2 or abatch with polypeptides comprising SEQ ID NO:1 and polypeptidescomprising SEQ ID NO:2), where the microparticles are formed by coolingthe sample from a temperature of about 25° C. to a temperature ofbetween about −45° C. to −55° C., the rate of cooling can be in therange of about between about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9or 1.0° C./min. In some embodiments, when the active agent is DAS181,the cooling rate is preset to a value of about 0.4° C./min or 0.5°C./min

The terms “ramping,” “controlled temperature ramping,” “controlledcooling,” “controlled rate of cooling” “controlled heating” or“controlled rate of heating” as used herein refer to heating or coolingsteps that are performed at a specified rate. “Holding” as used hereinrefers to a sample or reaction or composition being maintained at asteady temperature, within a range above or below the steady temperatureof 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.5, 1.7,2.0 or more upto about 2.5 or 3° C.

The term “gradual cooling” or “gradually cooling” or “gradually cooled”as used herein means that the rate at which the temperature of thefeedstock solution is lowered to a predetermined temperature at whichmicroparticles are formed can be subject to or determined by thetemperature differential at any given time between that of the feedstocksolution and the predetermined temperature to which it is being cooled.Thus, the rate of cooling can be variable and fluctuate during gradualcooling, as it is dependent on a temperature differential that canchange as cooling progresses, can generally vary between about 0.5° C.per minute and 15° C. per minute and typically is at least about 1° C.per minute.

The term “geometric standard deviation” or “GSD” as used herein is aterm of art that is a measure of uniformity of the size of themicroparticles in a formulation. A GSD of “1” means that allmicroparticles of the formulation have the same size, while a GSD of “5”generally indicates an un-uniform or polydisperse formulation. As usedherein, a “monodisperse” or “uniform” microparticle formulation meansthat the GSD of the formulation is between about 1.0-2.0, generallyabout 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7 or 1.8

The term “fine particle fraction” or “FPF,” as used herein, is a term ofart that is a measure of the fraction of particles of a microparticleformulation that are below a size considered suitable for deposition atthe site of interest. For example, in the case of microparticles to bedelivered to the central or upper respiratory tract, microparticles thatare greater than about 2 microns, preferably between about 3 microns toabout 10 microns are considered suitable for administration. This meansthat the FPF of the formulation below about 3.5, 3.4, 3.3, 3.2, 3.1,3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.0 or fewer microns generallyshould be low, of the order of 5%, 4%, 3%, 2%, 1%, 0.5% or less.

The term “particle size distribution” or “PSD” as used herein is a termof art that generally is an absolute measure of particle size and can bemeasured, for example, by laser diffraction (e.g., Sympatec HELOS LaserDiffraction System) or other methods known to those of skill in the art.In a laser diffraction-based method, the sizes of the particles of themicroparticle formulation are determined based on the differences in thepatterns of laser light diffraction generated by different sizedparticles. The diffracted light of a particular distribution ofintensity is collected by a multi-level photodetector and is convertedmathematically to extract information about the particle size.

The term “scavenger” or “scavenging agent” as used herein refers to acompound or composition that is a component of the microparticleformulations provided herein and protects the microparticle formulationsprovided herein from damage, degradation, aggregation, oligomerizationor any transformation that destabilizes the formulation or the activeagent in the formulation and/or decreases its activity over time. Insome embodiments, the destabilizing substances are present in thematerials used to package the microparticle formulations or are abyproduct arising during preparation of the materials used to packagethe formulations, such as aldehydes in hydroxypropyl methylcellulose(HPMC) capsules, pullulan polysaccharide capsules or laminate foilblister packs. In some embodiments, the scavenging agent can exert itsprotective effect by reacting with the destabilizing substance at a ratethat is faster than the rate of reaction between the active agent andthe destabilizing substance. In other embodiments, the scavenging agentcan protect the active agent from damage by forming a complex withgroups on the active agent that would otherwise react with thedestabilizing substances to form degradation products, aggregates,oligomers or other such products. Among the scavenging agents used inthe preparation of microparticle formulations are amines, generallyprimary or secondary amines, chelators, antioxidants, sugars orcombinations thereof.

B. Methods for Preparing Microparticle Formulations of Uniform Size

Provided herein are methods of producing uniform sized microparticleformulations of a macromolecule or small molecule active agent, whereinthe resulting microparticles are of a desired predetermined size andhave a narrow range of size distribution (geometric standard deviation,i.e., GSD of between 1.2-1.6). The methods provided herein include thesteps of mixing together a solution of an active agent in an aqueoussolvent, a counterion and an organic solvent and cooling the resultingmixture (also referred to herein as cocktail solution or feedstocksolution) to a predetermined temperature below about 25° C., generallybetween about −45° C. to about −60° C., at a cooling rate that ismaintained at a constant, preset and fixed value until the mixture is atthe predetermined temperature below about 25° C., whereby a compositioncontaining uniform sized microparticles of the active agent is formed.The resulting microparticles can be separated from unincorporatedcomponents in the feedstock solution by, for example, sedimentation,filtration and/or freeze-drying. The methods can further include stepsof holding the feedstock solution at the predetermined temperature(generally between about −45° C. to about −60° C.) at which themicroparticles are formed, then ramping up the temperature for primaryand optionally secondary drying steps, whereby volatiles present in themicroparticle formulations can be removed by sublimation. Severalcomponents and steps used in the methods provided herein, e.g., thetypes of active agents, counterions and organic solvents, cooling toform the microparticles, freeze-drying the resulting microparticles,have been described in detail elsewhere and are incorporated byreference herein (see, e.g., published U.S. Applications Serail Nos.20070190163 A1 and its continuation, 20100166874 A1, both of which aretitled “Technology for Preparation of Macromolecular Microparticles” andpublished U.S. Application Serial No. 20090098207 A1 titled “Technologyfor the Preparation of Microparticles”).

In the methods provided herein, microparticle formulations of a desiredsize can be obtained by choosing a cooling rate that can produceparticles of that size. The cooling rate can be determined empiricallyfor a given mixture of an active agent, counterion and organic solvent,using screening methods as described elsewhere (see, e.g., publishedU.S. Applications Serial Nos. 20070190163 A1 and its continuation,20100166874 A1, both of which are titled “Technology for Preparation ofMacromolecular Microparticles” and published U.S. Application Serial No.20090098207 A1 titled “Technology for the Preparation ofMicroparticles”). In general, a faster cooling rate produces smallerparticle sizes, while a slower cooling rate produces larger particlesizes. To obtain uniform formulations, the cooling rate can further bemaintained at a constant, fixed value as the feedstock solution iscooled from a temperature at or above 25° C. to a predeterminedtemperature below about 25 C (generally between about −45° C. to about−60° C.) whereby microparticles are formed.

The methods provided herein further produce microparticle formulationsthat are uniform or monodisperse, with a GSD of between 1.0-1.8,generally between about 1.2-1.6. The uniformity of the size is achievedby maintaining the cooling rate of the feedstock solution at a constant,fixed value, beginning at a temperature above or at about 25° C. untilthe mixture is cooled to the desired predetermined temperature belowabout 25° C. and microparticles are formed. The cooling rate can bemaintained at a fixed value by mechanical means, such as stirring thefeedstock solution to cool the solution at a specified preset rate, orby programming a computer to maintain a fixed cooling rate, regardlessof the difference in temperature between the feedstock solution at anygiven time during the cooling process and the final predeterminedtemperature to which it is cooled for microparticle formation.

For example, in the case of the sialidase fusion protein DAS181, whosesequence is set forth in SEQ ID NO:1 and SEQ ID:2, when the methodsemploy faster cooling rates and counterions such as sodium sulfate andmagnesium sulfate, smaller microparticles (value between about 3 micronsto about 8 microns mass median aerodynamic diameter (MMAD)) of DAS181(SEQ ID NO:1 or SEQ ID NO:2 or a batch with polypeptides comprising SEQID NO:1 and polypeptides comprising SEQ ID NO:2) are formed. When themethods employ slower cooling rates and counterions such as citrate,larger microparticles (value between about 8 microns to about 11 micronsMMAD) of DAS181 (SEQ ID NO:1 or SEQ ID NO:2 or a batch with polypeptidescomprising SEQ ID NO:1 and polypeptides comprising SEQ ID NO:2) areformed.

In particular embodiments, provided herein are methods of making uniformmicroparticle formulations of DAS181 (SEQ ID NO:1 or SEQ ID NO:2 or abatch with polypeptides comprising SEQ ID NO:1 and polypeptidescomprising SEQ ID NO:2) with a mass median aerodynamic diameter (MMAD)that has a value between about 3 microns to about 8 microns (e.g., 5-7microns or 6-7 microns) and a geometric standard deviation (GSD) ofbetween about 1.2 to about 2.0.

In some cases, e.g., acute parainfluenza in an immunocompromisedpatient, it is useful to treat with microparticles having a MMAD ofabout 3-8 microns (5.5-7.5 microns), which permits their deposition atin the lower portion of the respiratory tract, while avoiding depositionin the deep lung, e.g., alveoli and/or absorption into the blood stream,which could compromise their pharmacokinetic and/or safety profiles.

In the methods provided herein, the microparticle formulation of theprotein can also be tailored to a desired predetermined particle sizeand uniform size distribution by modifying one or more additionalparameters or steps, including one or more of the following: (1) theconcentration of the protein in the feedstock solution; (2) the natureand/or concentration of the counterion in the feedstock solution; (3)the nature and/or concentration of the organic solvent in the feedstocksolution; (4) the concentration of excipient; (5) the volume offeedstock solution in the lyophilization trays; and (6) one or morecontrolled temperature ramping (temperature decreasing and/ortemperature increasing) steps for forming and isolating the resultingmicroparticles.

The methods provided herein for making microparticle formulations caninclude additional steps. For example, after cooling the feedstocksolution at a constant, fixed rate to a predetermined temperature belowabout 25° C. for forming the microparticles, the cooled solution can beheld at that predetermined temperature for a specified period of time toremove unincorporated components of the microparticles by freeze-drying.The microparticles can further be heated in primary and optionallysecondary drying steps to remove volatile components by sublimation. Insome embodiments, the resulting microparticles are further packaged intomaterials containing formaldehyde and/or other aldehydes, such ashydroxypropyl methylcellulose (HPMC), aluminum foil laminates, gelcapsules or pullulan polysaccharide.

C. Uniform Microparticle Formulations

Also provided are uniform microparticle formulations prepared accordingto the methods provided herein. In some embodiments, the active agent orAPI in the formulation is for treating diseases that affect therespiratory tract such as influenza, asthma and COPD. An example of anAPI that can be used to treat respiratory diseases and disorders is thesialidase fusion protein, DAS181 (SEQ ID NO:1 or SEQ ID NO:2 or a batchwith polypeptides comprising SEQ ID NO:1 and polypeptides comprising SEQID NO:2). For administering DAS181 to the central and lower respiratorytract it is desirable to have uniform microparticle formulations withparticles of mass median aerodynamic diameter (MMAD) between about 3microns to about 8 microns and a GSD of between about 1.2 to 2.0. Ingeneral, particles with MMAD between about 3 microns to about 5 micronsare expected to deposit in the lower respiratory tract, particles withMMAD between about 5 microns to about 8 microns are expected to depositin the central to upper respiratory tract, and particles of about 8microns to about 10 or 11 microns are expected to deposit primarily inthe upper respiratory tract. Microparticles that are smaller than about2 or 2.5 microns, upon inhalation, can reach alveoli of the lungs andrelease the drug, where it can be absorbed into the bloodstream andcould compromise the therapeutic (pharmacokinetic) profile or safetyprofile of the drug. For such drugs, including DAS181, it is desirablethat particles smaller than about 2.0 or 2.5 microns are present at avery low level or are essentially absent from pharmaceuticalformulations not intended for pulmonary delivery or systemic absorption.

Thus, in some embodiments, provided herein are uniform-sizedmicroparticle formulations of DAS181 (SEQ ID NO:1 or SEQ ID NO:2 or abatch with polypeptides comprising SEQ ID NO:1 and polypeptidescomprising SEQ ID NO:2) or other active agents useful for preventing ortreating infections and other disorders of the respiratory tract such asinfluenza, parainfluenza, asthma and COPD, wherein the microparticlesare of a size that is suitable for deposition in the throat, trachea orbronchi. For optimal deposition of the microparticles at target sites ofthe infection or disorder (upper respiratory for influenza, centralrespiratory for asthma), the microparticles must not be (a) so big thatthey are trapped at the front end in the mouth (e.g., greater than about10.5 or 11 microns); or (b) so small that they are deposited deep in thelungs and absorbed systemically into the blood stream through thealveoli where they are not active and/or can be toxic (e.g., less thanabout 2.0 or 2.5 microns).

The microparticle formulations obtained by the methods provided hereinare of a uniform size distribution, i.e., monodisperse, with a geometricstandard deviation (GSD) of between about 1.2 and 2.0. Because of theiruniform or monodisperse character, the DAS181 (SEQ ID NO:1 or SEQ IDNO:2 or a batch with polypeptides comprising SEQ ID NO:1 andpolypeptides comprising SEQ ID NO:2) microparticle formulations providedherein, which generally are of a size between about 3 microns and about8 microns, have few to no particles less than about 2.5 microns, thusminimizing undesirable deposition of the drug into the deep lung and/orabsorption into the bloodstream. The microparticle formulations are alsohomogeneous, i.e., the formulation components are not segregated and areevenly distributed throughout the particles. The microparticleformulation may be homogenous, i.e., in the case of DAS181, the API canbe comprised of SEQ ID NO:1 or SEQ ID NO:2, or heterogeneous, i.e., inthe case of DAS181, the API can be comprised of a batch withpolypeptides comprising SEQ ID NO:1 and polypeptides comprising SEQ IDNO:2, with regard to the components of the API. Further, the fineparticle fraction (FPF) containing microparticles that are smaller than5 microns is less than 10%, generally less than about 8%, 7%, 6%, 5%,4%, 3.5%, 3%, 2.5%, 2%, 1.5% or 1%. In one embodiment, a microparticleformulation of DAS181 (SEQ ID NO:1 or SEQ ID NO:2 or a batch withpolypeptides comprising SEQ ID NO:1 and polypeptides comprising SEQ IDNO:2) for the prophylaxis or treatment of influenza has a mass medianaerodynamic diameter (MMAD) of 5-8 (6-7) microns, a GSD of 1.4-1.6, anFPF of less than 5% (2-4%) for particle sizes less than 5 microns.

D. Methods of Making Microparticle Formulations that are Stable in theirPackaging

Also provided herein are methods of making stable microparticleformulations and the resulting stable microparticle formulations inwhich the active agent (or API, for a pharmaceutical formulation) isprotected from degradation or aggregation resulting from materialspresent in the packaging container or delivery system. The methodsprovided herein include the steps of:

mixing together: (i) a solution of an active agent in an aqueoussolvent, (ii) a counterion selected from among citric acid/citrate,magnesium sulfate, potassium sulfate or calcium sulfate, phosphate,pivalate, rubidium, bromine, perchlorate, itaconate, and any salt, acid,or base form thereof, (iii) one or more scavenging agents and (iv) anorganic solvent; and

cooling the resulting mixture (also referred to herein as cocktailsolution or feedstock solution) to a predetermined temperature belowabout 25° C. either gradually or at a cooling rate that can bemaintained at a constant fixed value until the mixture is at thepredetermined temperature below about 25° C., whereby a compositioncontaining microparticles that include the active agent at about 50% toabout 85% wt/wt, the counterion at about 2% to about 6% wt/wt and thescavenging agents at about 8% to about 40% wt/wt is formed. Theresulting microparticles can be separated from the mixture to removecomponents other than the microparticles by, for example, sedimentation,filtration and/or freeze-drying.

The resulting dry microparticles, in certain embodiments, can have acomposition of between about 50% to about 75% wt/wt of the active agent,between about 2% to about 6% wt/wt of the counterion, between about 5%to about 40% wt/wt of each scavenging agent and between about 5% toabout 10% residual moisture. The scavenging agent can be a primary orsecondary amine, a chelator, an antioxidant, a sugar, or combinationsthereof and generally is present in at least a 100-fold excess, uptoabout a 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750,800, 850, 900, 950 or a 1000 fold or more molar excess relative to thealdehyde(s) present in the material used to package the microparticles.The scavenging agent is incorporated into the microparticles obtained bythe methods provided herein, and can protect the active agent againstthe damaging effects of some materials, such as aldehydes, that may bepresent in a packaging container or delivery system such as HPMCcapsules, certain gel capsules, pullulan polysaccharide capsules andaluminum foil laminate blister packs.

In some embodiments of the method, the active agent is a protein and inparticular embodiments, the protein is DAS181 (SEQ ID NO:1 or SEQ IDNO:2 or a batch with polypeptides comprising SEQ ID NO:1 andpolypeptides comprising SEQ ID NO:2). In other embodiments, thescavenging agent is histidine. In yet other embodiments, the scavengingagent is tryptophan. In further embodiments, combinations of scavengingagents, such as an amino acid and a sugar, more than one amino acid, ormore than one amino acid and a sugar, are used; in particularembodiments, the combinations of scavenging agents are histidine andtrehalose, histidine and sucrose, glycine and sucrose, histidine,glycine and sucrose, or histidine and tryptophan. In some embodiments,the microparticle formulations containing an active agent, a counterionand a scavenging agent as provided herein can further be tailored to adesired predetermined particle size and uniform size distribution byapplying a constant, fixed cooling rate to the feedstock solution forcooling the feedstock solution from a temperature above or at 25° C. toa predetermined temperature below 25° C., whereby microparticles areformed. Additional parameters or steps that can be used to obtain adesired particle size and/or size distribution can include one or moreof the following: (1) the concentration of the active agent in thefeedstock solution; (2) the nature and/or concentration of thecounterion in the feedstock solution; (3) the nature and/orconcentration of the organic solvent in the feedstock solution; (4) theconcentration of excipient; (5) the volume of feedstock solution in thelyophilization trays; and (6) one or more controlled temperature ramping(temperature decreasing and/or temperature increasing) steps for formingand isolating the resulting microparticles.

Also provided herein are microparticles of a protein containing betweenabout 50% to about 85% wt/wt of the protein, about 2% to about 6% wt/wtof a counterion selected from among citric acid/citrate, magnesiumsulfate, potassium sulfate or calcium sulfate, phosphate, pivalate,rubidium, bromine, perchlorate, itaconate, and any salt, acid, or baseform thereof, and about 8% to about 40% wt/wt of one or more scavengers.In some embodiments, the protein component of the microparticles can becomprised of a single first polypeptide. In some embodiments, theprotein component of the microparticles can be comprised of a single yetdifferent second polypeptide and being the same protein as the firstpolypeptide. In another embodiment, the protein component of themicroparticles can be comprised of a composition of the firstpolypeptide and the second polypeptide. In one embodiment, the proteinis DAS181 (SEQ ID NO:1 or SEQ ID NO:2 or a batch with polypeptidescomprising SEQ ID NO:1 and polypeptides comprising SEQ ID NO:2) and infurther embodiments, the scavenging agent is present in about 9% toabout 11% wt/wt of the dry powder microparticles. In some embodiments,the scavenging agent(s) present in the DAS181 microparticle formulationsis histidine, histidine/histidine HCl, histidine/trehalose,histidine/tryptophan, histidine/sucrose, glycine/sucrose, orhistidine/glycine/sucrose.

In the methods provided herein, the microparticle formulation of theprotein can also be tailored to a desired predetermined particle sizeand uniform size distribution by modifying one or more additionalparameters or steps, including one or more of the following: (1) theconcentration of the protein in the feedstock solution; (2) the natureand/or concentration of the counterion in the feedstock solution; (3)the nature and/or concentration of the organic solvent in the feedstocksolution; (4) the concentration of excipient; (5) the volume offeedstock solution in the lyophilization trays; and (6) one or morecontrolled temperature ramping (temperature decreasing and/ortemperature increasing) steps for forming and isolating the resultingmicroparticles.

The methods provided herein for making microparticle formulations caninclude additional steps. For example, after cooling the feedstocksolution at a constant, fixed rate to a predetermined temperature belowabout 25° C. for forming the microparticles, the cooled solution can beheld at that predetermined temperature for a specified period of time toremove unincorporated components of the microparticles by freeze-drying.The microparticles can further be heated in primary and optionallysecondary drying steps to remove volatile components by sublimation. Insome embodiments, the resulting microparticles are further packaged intomaterials containing formaldehyde and/or other aldehydes, such ashydroxypropyl methylcellulose (HPMC), aluminum foil laminates, gelcapsules or pullulan polysaccharide.

E. Microparticle Formulations that are Stable in their Packaging

Also provided herein are microparticle formulations that are packaged inmaterials containing formaldehyde and/or other aldehydes. Such materialsinclude, but are not limited to, hydroxypropyl methylcellulose (HPMC)capsules, certain gel capsules, aluminum foil laminate blister packs andpullulan polysaccharide capsules.

Also provided herein are articles of manufacture that contain amicroparticle formulation that includes a sialidase or a sialidasefusion protein in an amount of about 60% to about 75% wt/wt, acounterion and a scavenging agent that is a primary amine in the amountof about 8% to about 11% wt/wt, a packaging material for theformulation, where the material contains formaldehyde and/or otheraldehydes, and a label that indicates that the composition is for atherapeutic indication. In one embodiment, the therapeutic indication isinfluenza. In other embodiments, the therapeutic indication is asthma orCOPD. In yet other embodiments, the therapeutic indication is selectedfrom among parainfluenza, RSV, sinusitis, otitis, laryngitis,bronchitis, pneumonia, bronchiectasis, vasculitis, mucous plugging,Wegener's granulomatosis and cystic fibrosis (CF). In some embodiments,the scavenging agent is histidine; in other embodiments, the scavengingagent is a combination of histidine and trehalose. In yet otherembodiments, the counterion is selected from among citric acid/citrate,magnesium sulfate, potassium sulfate or calcium sulfate, phosphate,pivalate, rubidium, bromine, perchlorate, itaconate, and any salt, acid,or base form thereof. The packaging material can be a HPMC capsule; infurther embodiments, the HPMC capsule is clear. In other embodiments,the packaging material can be a gel capsule, or a pullulanpolysaccharide capsule. In yet other embodiments, the article ofmanufacture further contains a secondary packaging material; in someembodiments, the secondary packaging material is a foil laminate; inparticular embodiments, the foil laminate is a cold form foil aluminumlaminate blister pack. In some embodiments, the scavenging agent is anamine; in further embodiments, the amine is selected from among lysine,histidine, glycine, arginine, glutamine, glutamic acid, cysteine,alanine, tyrosine, tryptophan, aminoguanidine, cysteamine, serine,carnosine, hydralazine and poly(l-lysine). In one embodiment, thesialidase fusion protein is DAS181 having the sequence set forth in SEQID NO:1 or SEQ ID NO:2 or a batch with polypeptides comprising SEQ IDNO:1 and polypeptides comprising SEQ ID NO:2; in a particularembodiment, the sialidase fusion protein is DAS181 having the sequenceset forth in SEQ ID NO:1 or SEQ ID NO:2 or a batch with polypeptidescomprising SEQ ID NO:1 and polypeptides comprising SEQ ID NO:2 and thescavenging agent is histidine or a combination of histidine andtrehalose.

The articles of manufacture provided herein also can contain an inhalerfor pulmonary administration of the composition. In certain embodiments,the inhaler is a dry powder inhaler, a metered dose inhaler or anelectrostatic delivery device.

The microparticles obtained by the methods provided herein are useful asprophylactic, therapeutic or diagnostic agents for treating ordiagnosing disease states in a subject in vivo or in vitro.

Active Agents

In some embodiments of the methods and formulations provided herein, theactive agents are proteins, including therapeutic proteins such asDAS181 (the sialidase fusion protein having the sequence of amino acidresidues set forth in SEQ ID NO:1 or SEQ ID NO:2 a mixture thereof).

Microparticle formulations of other sialidase fusion proteins, besidesDAS181, also are contemplated herein. Sialidase-GAG fusion proteins suchas DAS181 are proteins that are made up of a sialidase protein, orcatalytically active portion thereof, fused to a glycosaminoglycan(GAG)-binding sequence. As such, these proteins effectively contain ananchoring domain (the GAG-binding sequence) and a therapeutic domain(the sialidase protein, or catalytically active portion thereof). Thesialidase-GAG fusion proteins are designed to bind to the epithelium andremove the surrounding sialic acids, and can therefore be used as atherapeutic agent against pathogens that utilize sialic acids in theinfection process. The ability of the fusion protein to bind to theepithelium increases its retention when the fusion protein isadministered, for example, as an inhalant to treat influenza infection.The GAG-binding sequence acts as an epithelium-anchoring domain thattethers the sialidase to the respiratory epithelium and increases itsretention and potency.

Counterion

The selection and characterization of counterions has been describedextensively elsewhere and is incorporated by reference herein (see,e.g., published U.S. Applications Serial Nos. 20070190163 A1 and itscontinuation, 20100166874 A1, both of which are titled “Technology forPreparation of Macromolecular Microparticles” and published U.S.Application Serial No. 20090098207 A1 titled “Technology for thePreparation of Microparticles”). The counterions magnesium sulphate andcitric acid or citrate, when used in the methods provided herein, canproduce microparticles that are of a size that is suitable foradministration of a drug to the respiratory tract while avoidingsignificant absorption into the bloodstream.

Nature and Concentration of Organic Solvent

An organic solvent added to the cocktail in the methods provided hereingenerally is not a polymer, generally can be water miscible and isselected from among alcohols as described elsewhere and incorporated byreference herein (see, e.g., published U.S. Applications Serial Nos.20070190163 A1 and its continuation, 20100166874 A1, both of which aretitled “Technology for Preparation of Macromolecular Microparticles” andpublished U.S. Application Serial No. 20090098207 A1 titled “Technologyfor the Preparation of Microparticles”). In some embodiments of themethods provided herein, the organic solvent is isopropanol. In general,the organic solvent isopropanol is a good solvent of choice because (1)it is a class 3 solvent (i.e., safe), (2) it can produce microspheres ina wide range (2-30%, v/v) of concentrations, and (3) it has a relativelyhigh freezing point so its vapors can efficiently be trapped duringlyophilization. In particular embodiments of the methods providedherein, the final concentration of isopropanol is 25% or 26%.

Cooling Ramp

The feedstock solutions from which microparticles are formed accordingto the methods provided herein are cooled at a constant, fixed presetrate—beginning at a temperature of above or at 25° C. at which thefeedstock solution initially is present, and ending at a predeterminedtemperature below about 25° C. at which the microparticles are formed.The predetermined temperature at which microparticles are formed isempirically determined based on the type of macromolecule, solvents,counterions and other ingredients as well as the rate of cooling and canvary from about or at 15° C., 10° C., 8° C., 5° C., 3° C., 2° C., 1° C.,−2° C., −5° C., −7.5° C., −10° C., −15° C., −20° C., −25° C., −30° C.,−35° C., −40° C., −45° C., −50° C. or −55° C.

The rate at which cooling and freezing of the cocktail (cooling ramp) isperformed can determine the final size of the microparticles. Ingeneral, a faster cooling ramp yields smaller microparticles whereas aslower cooling ramp yields larger microparticles. In the methodsprovided herein, the cooling rates generally are selected to producemicroparticles that are larger than about 3 microns and smaller thanabout 11 microns. Depending on the size of microparticles desired andthe type of active agent, the cooling rate can be from about 0.01°C./min to about 1° C./min. In general, the cooling rate is less than 1°C./min and is about 0.3, 0.4, 0.5, 0.6, 0.7 or 0.8° C./min. In someembodiments, the cooling rate is 0.4 C/min or 0.5 C/min.

Scavenging Agents

Microparticle formulations of several active agents or drugs containingamine groups, exemplary of which is the protein DAS181, often arepackaged in containers that have components, such as aldehydes, whichcan react with amine groups present on the active agents, therebyforming cross-linked aggregates and affecting their activity. Exemplarypackaging materials that are known to contain aldehydes, eitherinherently or as a by-product of their formation include hydroxypropylmethyl cellulose (HPMC) capsules and aluminum foil laminates. Themanufacture of HPMC includes a step of methyl cellulose reacting withpropylene oxide to produce hydroxypropyl methyl cellulose. Without beingbound by any theory, during this process, propylene oxide can react withhydroxide to form formaldehyde and acetaldehyde. The aldehydes, asvolatile compounds, can leach from HPMC capsules and contact theencapsulated materials (e.g., an API like DAS181). Upon contact, thealdehydes, even when present in trace amounts, (e.g., 10-20 ppm per HPMCcapsule) can react with amine groups of the API, resulting inmodification and crosslinking of the API and compromising its activityover time. The damaging effect of aldehydes may be enhanced (catalyzed)by other compounds/conditions present in the product and/or packagingsuch as certain metals (counterions or excipients), moisture andtemperature. Therefore, provided herein are methods of makingmicroparticle formulations in which the crosslinking/aggregating effectof aldehydes on the pharmaceuticals/nutraceuticals is reduced oreliminated. In embodiments of the method, the active agent is a protein;in some embodiments, the protein is selected from among sialidases,sialidase fusion proteins, proteases, protease inhibitors, cytokines,insulin, BSA, human growth hormone, calcitonin, recombinant human DNase,interferons and parathyroid hormone; in yet other embodiments, theprotein is a sialidase fusion protein. In a particular embodiment, thesialidase fusion protein is DAS181 having the sequence set forth in SEQID NO:1. In another particular embodiment, the sialidase fusion proteinis DAS181 having the sequence set forth in SEQ ID NO:2. In anotherparticular embodiment, the sialidase fusion protein is DAS181 and ispresent as a batch with polypeptides comprising SEQ ID NO:1 andpolypeptides comprising SEQ ID NO:2. In yet another particularembodiment, the sialidase fusion protein is DAS181 and the scavengingagent is histidine. In further embodiments, the sialidase fusion proteinis DAS181 and the scavenging agent is a mixture of histidine andtrehalose, histidine and tryptophan, tryptophan alone, glycine alone,glycine and sucrose, glycine, histidine and sucrose, or histidine andsucrose.

Other exemplary drugs or active agents containing amine groups that canbe shielded from packaging material (aldehyde)-mediated cross-linkingand/or degradation by forming drug microparticles containing ascavenging agent include, but are not limited to, sialidases, sialidasefusion proteins, proteases, protease inhibitors, cytokines, insulin,BSA, human growth hormone, calcitonin, recombinant human DNase,interferons, parathyroid hormone, exenatide-4, α-synuclein and knownsmall molecule drugs such as the proton pump inhibitor Nexium®(esomeprazole magnesium; Astra Zeneca), the antiviral Valtrex®(valacyclovir hydrochloride; Glaxo SmithKline), the anticonvulsantLyrica® (pregabalin; Pfizer), the anti-inflammatory drug Asacol®(mesalamine; Proctor & Gamble), the antihistamine Clarinex®(desloratadine; Schering Plough), the dopamine agonist Mirapex®(pramipexole; Boehringer Ingelheim) and the antiviral Zovirax®(acyclovir; Glaxo SmithKline).

The methods provided herein for stabilization of the active agents(pharmaceuticals/nutraceuticals) against the damaging effect ofaldehydes include adding a scavenging agent to the feedstock solutionwhereby the scavenging agent is incorporated into the resultingmicroparticle formulations containing the active agents. Exemplaryscavenging agents include, but are not limited to, primary and secondaryamines, chelators, antioxidants, sugars and combinations thereof.

In some embodiments, the scavenging agent is a primary or secondaryamine. Without being bound by any theory, the amine could act as ascavenging agent by reacting with the aldehyde, thereby protecting theamine groups of the active agent from reacting with the aldehyde. Infurther embodiments, the primary or secondary amine is selected fromamong lysine, histidine, glycine, arginine, glutamine, glutamic acid,cysteine, alanine, tyrosine, tryptophan, aminoguanidine, cysteamine,serine, carnosine, hydralazine and poly(l-lysine). In yet otherembodiments, the amine is histidine. Without being bound by any theory,the reaction of histidine with an aldehyde such as formaldehyde can formthe product spinacine, which is known to be non-toxic and therefore isless likely to pose a health risk when administered in a microparticleformulation containing an API. In exemplary microparticle formulationscontaining an amine as a scavenging agent, the concentrations of theamino acids in the dry powder are between about 0.5% and about 20% w/w.

In other embodiments, the scavenging agent is an antioxidant. Exemplaryantioxidants that can inhibit oligomer formation and destabilization ofproteins such as DAS181 include, but are not limited to, ascorbic acidand its derivatives (such as substituted compounds at 2-, 3-, 5- and6-positions including L-ascorbate-2-sulphate andL-ascorbate-2-phosphate, L-ascorbyl-6-palmitate), thioglycerol,glutathione, tocopherol, melatonin, sodium bisulfite, urea, ethyleneurea, 3,4-diaminobenzoic acid, allantoin, glycouril, anthranilic acid,methyl anthranilate, methyl 4-aminobenzoate, ethyl acetoacetate,acetoacetamide, malonamide, 1,3-dihydroxyacetone dimer, biuret, oxamide,benzoguanamine, pyroglutamic acid, pyrogallol, methyl gallate, ethylgallate, propyl gallate, triethanol amine, succinamide, thiabendazole,benzotriazol, triazole, indoline, sulfanilic acid, oxamide, glucose,cellulose, poly(vinyl alcohol), partially hydrolyzedpoly(vinylformamide), poly(vinyl amine), poly(ethylene imine),poly(oxyalkyleneamine), poly(vinyl alcohol)-co-poly(vinyl amine),poly(4-aminostyrene), poly(l-lysine), chitosan, hexane diol,ethylenediamine-N,N′-bisacetoacetamide, N-(2-ethylhexyl)acetoacetamide,2-benzoylacetoacetamide, N-(3-phenylpropyl)acetoacetamide, lilial,helional, melonal, triplal, 5,5-dimethyl-1,3-cyclohexanedione,2,4-dimethyl-3-cyclohexenecarboxaldehyde,2,2-dimethyl-1,3-dioxan-4,6-dione, 2-pentanone, dibutyl amine, ammoniumhydroxide, benzylamine, hydroxycitronellol, cyclohexanone, 2-butanone,pentane dione, dehydroacetic acid and mixtures thereof.

In the case of ascorbate derivatives, when the active agent is aprotein, it is thought that these compounds react with ε-amino groups ofL-lysine residues on the protein, thereby reducing the number of activesites on the protein that can react with aldehydes such as formaldehydethat are present in the packaging container/capsule. The ascorbatederivatives can also contain amino groups, thereby providing anadditional mechanism for reacting with the aldehydes and protecting theactive agents from their damaging effects. Exemplary derivatives thatinclude amine functionalities include L-ascorbic acid derivatives areselected from the group consisting of ascorbyl-6-lysine,ascorbyl-2-lysine, ascorbyl-6-polylysine, ascorbyl-2,6-dilysine,ascorbyl-6-polylysine-2-lysine, ascorbyl-6-lysine-2-polylysine,ascorbyl-2,6-polylysine, ascorbyl-6-proline, ascorbyl-2-proline,ascorbyl-6-polyproline, ascorbyl-2-polyproline, ascorbyl-2,6-diproline,ascorbyl-2-proline-6-polyproline, ascorbyl-2-polyproline-6-proline,ascorbyl-2,6-diproline, 6-deoxyascorbyllysine, 6-deoxyascorbylproline,6-deoxyascorbylpolylysine, 6deoxyascorbylpolyproline,6-deoxyascorbyllysine-2-proline, 6-deoxyascorbylproline-2-lysine,6-deoxyascorbylpolylysine-2-proline,6-deoxyascorbylpolyproline-2-lysine,6-deoxyascorbyllysine-2-polyproline,6-deoxyascorbylproline-2-polylysine, 6-deoxyascorbateproline-2-lysine-proline, 6-deoxyascorbate-2-proline-lysine,6-deoxyascorbyllysine, 6-deoxyascorbate-lysine-proline,6-deoxyascorbyl-lysine-2-proline, 6-deoxyascorbyl-polylysine-2-proline,6-deoxyascorbyl-lysine-2-polyproline,6-deoxyascorbyl-lysine-2lysine-proline, 6-deoxyaminoascorbyl-polylysine, 6-deoxyamino ascorbyl-lysine-proline, 6-deoxyaminoascorbylproline and 6-deoxyamino ascorbylpolyproline.

In yet other embodiments, the scavenging agent is a chelator. Anexemplary chelator is triethylenetetramine (TETA), which can beefficacious in preventing protein oligomer formation due to the presenceof four amine groups that can act as formaldehyde/aldehyde scavenger ora chelator. Histidine or tryptophan could also function as chelators.Other chelators that can act as scavenging agents in concert withprimary and/or secondary amines include diethylenetriamine pentaaceticacid (DTPA) and metal-based (e.g., copper, iron, manganese) chelatingagents

In further embodiments, sugars added to the microparticle formulationsprovided herein can inhibit the active agent oligomerization/aggregationthat can occur in the presence of aldehydes such as formaldehyde.Without being limited by any particular mechanism, when the active agentis a protein such as DAS181, the reduction of aggregation in presence ofsugar could be attributed to the sugars forming hydrogen-bonds with theavailable sites on the protein surface, thereby reducing access of thealdehydes to the amine groups on the protein. Sugars such as trehaloseand sucrose may also stabilize the tertiary structure of the protein,thereby providing better stability against thermal activation. The finalconcentrations of sugars in the dry microparticle formulations can bebetween about 1% and about 15% w/w. Thus, in some embodiments of themethods provided herein, a combination of a primary amine, such ashistidine, and a sugar, such as trehalose, are added to the feedstocksolution for incorporation into the microsphere formulations to provideadded stability to the formulations.

Optional Additional Agents

The compositions provided herein can optionally, in addition to anactive agent, contain one or more surfactant and/or additionalpharmaceutical or nutraceutical or other such agent for ingestion by asubject; such additional agents and their characteristics andproportions in the microparticle formulations are described extensivelyin U.S. Applications Serial Nos. 20070190163 A1 and its continuation,20100166874 A1, both of which are titled “Technology for Preparation ofMacromolecular Microparticles” and in published U.S. Application SerialNo. 20090098207 A1 titled “Technology for the Preparation ofMicroparticles,” all of which are incorporated by reference herein.

F. Uses of the Compositions

Therapeutic and diagnostic applications of the microparticles caninclude drug delivery, vaccination, gene therapy, and in vivo tissue ortumor imaging. Routes of administration can include oral or parenteraladministration; mucosal administration; ophthalmic administration;intravenous, subcutaneous, intra-articular, or intramuscular injection;inhalation administration; and topical administration. In particularembodiments, the microparticles are suitable for pulmonaryadministration by inhalation for the prevention, prophylaxisor treatmentof diseases of the respiratory tract including influenza, parainfluenza,RSV, sinusitis, otitis, laryngitis, bronchitis, pneumonia, allergic andnon-allergic asthma, COPD, bronchiectasis, vasculitis, mucous plugging,Wegener's granulomatosis and cystic fibrosis (CF).

The microparticle formulations provided herein can be formulated astablets, caplets, gels, vials, pre-filled syringes, inhalers,electrostatic devices and other devices for delivery. In someembodiments, the packaging materials used to package the formulationscontain formaldehyde or other aldehydes; exemplary materials includeHPMC, certain gels, aluminum foil laminates and pullulanpolysaccharides. The delivery dosage of the formulations can be frombetween about 0.5 mg protein per dose to about 100 mg protein per dose,or about 0.75 mg, 1 mg, 1.5 mg, 2 mg, 3 mg, 5 mg, 10 mg, 15 mg, 20 mg,30 mg, 40 mg, 45 mg, 50 mg, 55 mg or 60 mg protein per dose. Thefrequency and amount of administration of a dose, for example, for thetreatment or prophylaxis of influenza or asthma using DAS181, can befrom three or more times a day, to two times a day, to once a day, totwo times a week, to once a week, to once every two weeks or lessfrequent than once every two weeks. In some embodiments for thetreatment of influenza, parainfluenza or asthma, the delivery dose of aDAS181 (SEQ ID NO:1 or SEQ ID NO:2 or a batch with polypeptidescomprising SEQ ID NO:1 and polypeptides comprising SEQ ID NO:2)microparticle formulation by inhalation is 10 mg; in particularadministrations, 13 mg of Formulation A filled in a #3 clear HPMCcapsule (delivered dose of 10 mg) is administered once a day for thetreatment of influenza, parainfluenza or asthma, and in furtherembodiments the administration can be for up to 3 consecutive days.

Packaging of the Dry Powder

The dry microparticle formulation was packaged in a primary containerclosure system that is a natural (clear), Size 3, HPMC capsule(Capsugel). The dry powder fill mass for each capsule was about 11 mg toachieve a 10 mg delivered dose. The packaged HPMC capsules were then putinto a cold form aluminum laminate blister pack as a secondary containerclosure system

Example 1

Manufacture of Uniform Stable Microparticles of DAS181 with MagnesiumSulfate as the Counterion and Histidine as the Scavenging Agent

A. Purification of DAS181

DAS181 is a fusion protein containing the heparin (glysosaminoglycan, orGAG) binding domain from human amphiregulin fused via its N-terminus tothe C-terminus of a catalytic domain of Actinomyces Viscosus (e.g., SEQID NO: 1 (without amino terminal methionine) and SEQ ID NO: 2 (withamino terminal methionine)). The DAS181 protein used in the examplesbelow was purified as described in Malakhov et al., Antimicrob. AgentsChemother., 1470-1479 (2006), which is incorporated in its entirety byreference herein. Briefly, the DNA fragment coding for DAS181 was clonedinto the plasmid vector pTrc99a (Pharmacia) under the control of a IPTG(isopropyl-β-D-thiogalactopyranoside)-inducible promoter. The resultingconstruct was expressed in the BL21 strain of Escherichia Coli (E.Coli). The E. coli cells expressing the DAS181 protein were washed bydiafiltration in a fermentation harvest wash step using Toyopearl buffer1, UFP-500-E55 hollow fiber cartridge (GE Healthcare) and aWatson-Marlow peristaltic pump. The recombinant DAS181 protein was thenpurified in bulk from the cells as described in published U.S.Applications Serial Nos. 20050004020 A1 and 20080075708 A1, which areincorporated in their entirety by reference herein.

B. Activity of DAS181

The sialidase activity of DAS181 was measured using the fluorogenicsubstrate 4-methylumbelliferyl-N-acetyl-α-D-neuraminic acid (4-MU-NANA;Sigma). One unit of sialidase is defined as the amount of enzyme thatreleases 10 nmol of MU from 4-MU-NANA in 10 minutes at 37° C. (50 mMCH₃COOH—NaOH buffer, pH 5.5) in a reaction that contains 20 nmol of4-MU-NANA in a 0.2 ml volume (Potier et al., Anal. Biochem., 94:287-296,1979). The specific activity of DAS181 was determined to be 1,300 U/mgprotein (0.77 μg DAS181 protein per unit of activity).

C. Batch Mode Process and Formulation of DAS181 Microparticles

The following ingredients were combined to form DAS181 microparticles ina large scale batch process:

-   -   (a) 75 mg/ml Histidine, pH 6.0, 1 M Trehalose, 1.25 M magnesium        sulfate and 100 mM calcium chloride stock solutions were sterile        filtered into and combined in an Excipient Bottle.    -   (b) The contents of the Excipient Bottle were added, with        mixing, to a Compounding Vessel containing 125 mg/ml DAS181        protein prepared as described above.    -   (c) Isopropanol was sterile filtered into an Isopropanol Bag    -   (d) The content of the Isopropanol Bag was pumped into the        Compounding Vessel while mixing vigorously to form the Feedstock        Solution. The final composition of the Feedstock Solution was as        follows: 70 mg/ml DAS181, 25% isopropanol, 10.5 mg/ml histidine,        10.5 mg/ml trehalose, 5.06 mg/ml magnesium sulfate, 0.19 mg/ml        calcium chloride, pH 6.0. The time between initiating the        addition of isopropanol and starting the lyophilization cycle        was between 90 minutes and 120 minutes    -   (e) Stainless Steel trays that had undergone depyrogenation were        each filled with 950 g of the Feedstock Solution, using a        metering pump    -   (f) The filled Stainless Steel trays were subsected to a        Lyophilization Cycle as follows:        -   a. the trays were gasketed and placed in the lyophilizer            shelves at 25° C. for 5 minutes;        -   b. the temperature of the shelves was lowered to −55° C. at            a ramp rate of −0.5° C./minute;        -   c. the trays were held at −55° C. for between 60 and 180            minutes;        -   d. primary drying was accomplished by setting the condenser            to <−60° C., applying a vacuum of 125 mTorr and increasing            the temperature to −40° C. at a ramp rate of 0.125°            C./minute and further to a temperature of −25° C. at 0.25°            C./minute;        -   e. the temperature was held at −25° C. for between 5000 and            6500 minutes;        -   f. secondary drying was accomplished by increasing the            temperature to 15° C. at a ramp rate of 0.67° C./minute and            further to a temperature of 30° C. at a ramp rate of 0.5°            C./minute;        -   g. the temperature was held at 30° C. for between 300 and            500 minutes; and        -   h. the vacuum was released and the lyophilizer was            backfilled with nitrogen to prevent oxidation of the            microparticle formulation before transferring into bottles            for bulk mixing and aliquoting the bulk powder for storage            at <−15° C.            Transfer of Bulk DAS181 Microparticles into Container and            Mixing

A section on the bottom film of each Stainless Steel tray was cleanedusing sanitizing wipes and a 3×3 cm opening was made with a scalpel. Thedry microparticles were transferred into a plastic bottle. The bottlewas capped and tumbled forty times, changing directions with eachinversion. The tumbling was to ensure uniformity of bottle content.Samples for analytical testing were taken and the bottle was recappedand sealed into plastic bags for storage at ≤−15° C.

Dry Powder Composition and Properties (Formulation II Example)

The DAS181 microparticle formulation dry powder, prepared according tothe method described above as the following composition and physicalparameters:

Composition (wt/wt %):

DAS181: 64.5-64.7%

Histidine free base: 4.3-4.6%

Histidine HCl: 5.9-6.3%

Trehalose: 9.1-9.7%

Magnesium sulfate: 4.7-5.8%

Calcium chloride: 0.2%

Sodium acetate (trace amounts from DAS181 stock solution): 0.04-0.05%

Acetic acid (trace amounts from DAS181 stock solution): 0.02%

Water: 10%

Isopropanol: trace amounts

Physical Parameters:

The DAS181 dry powder microparticles prepared according to the abovemethod have a mass median aerodynamic diameter (MMAD) of 6.4 microns, aGSD of 1.4-1.6, an FPF of 0.9-2.1% for particle sizes<3.2 microns and anFPF of 8.9-10.7% for particle sizes<5 microns.

The suitability of the microparticles for administration by oralinhalation to treat respiratory tract infections such as influenza wastested by Andersen Cascade Impaction. The deposition of pharmaceuticalsin the respiratory tract can be predicted by deposition of particles(microparticles) on the stages/collection plates of the cascadeimpactor. For the DAS181 microparticles, when administered to prevent ortreat viral infections that initiate in the respiratory tract, such asinfluenza, it is desirable to deposit the drug in the throat, tracheaand bronchi (upper respiratory airway) while avoiding deposition at thesecondary and terminal bronchi and the alveoli. The results showed thatonly about 1.2% of the microparticles were deposited at the collectionplates corresponding to secondary and terminal bronchi and the alveoli,with about 0.2% being deposited at the alveoli.

Packaging of the Dry Powder

The dry microparticle formulation was packaged in a primary containerclosure system that is a natural (clear), Size 3, HPMC capsule(Capsugel). The dry powder fill mass for each capsule was about 13 mg toachieve a 10 mg delivered dose. The packaged HPMC capsules were then putinto a cold form aluminum laminate blister pack as a secondary containerclosure system

Example 2

Manufacture of Uniform Stable Microparticles of DAS181 with CitricAcid/Citrate as the Counterion and Histidine as the Scavenging Agent

The DAS181 protein was purified and its activity measured as describedin Example 1 (Sections 1A and 1B). The following ingredients were thencombined to form DAS181 microparticles in a large scale batch process:

-   -   (a) 75 mg/ml Histidine, 0.107M citric acid, pH 5.0 and 1M        Trehalose stock solutions were sterile filtered into and        combined in an Excipient Bottle.    -   (b) The contents of the Excipient Bottle were added, with        mixing, to a Compounding Vessel containing 125 mg/ml DAS181        protein prepared as described in Example 1.    -   (c) Isopropanol was sterile filtered into an Isopropanol Bag    -   (d) The content of the Isopropanol Bag was pumped into the        Compounding Vessel while mixing vigorously to form the Feedstock        Solution. The final composition of the Feedstock Solution was as        follows: 70 mg/ml DAS181, 26% isopropanol, 9.8 mg/ml histidine,        9.8 mg/ml trehalose, 2.69 mg/ml citric acid, pH 5.0. The time        between initiating the addition of isopropanol and starting the        lyophilization cycle was between 90 minutes and 120 minutes    -   (e) Stainless Steel trays that had undergone depyrogenation were        each filled with 950 g of the Feedstock Solution, using a        metering pump    -   (f) The filled Stainless Steel trays were subjected to a        Lyophilization Cycle as follows:        -   a. the feedstock solution in the lyophilization trays were            gasketed and placed in the lyophilizer shelves at 25° C. for            5 minutes;        -   b. the temperature of the shelves was lowered to −55° C. at            a ramp rate of −0.4° C./minute;        -   c. the trays were held at −55° C. for between 60 and 180            minutes;        -   d. primary drying was accomplished by setting the condenser            to <−60° C., applying a vacuum of 125 mTorr with 250 mTorr            dead band and increasing the temperature to −40° C. at a            ramp rate of 0.125° C./minute and further to a temperature            of −30° C. at 0.167° C./minute;        -   e. the temperature was held at −30° C. for between 5000 and            6500 minutes;        -   f. secondary drying was accomplished by increasing the            temperature to 15° C. at a ramp rate of 0.5° C./minute,            holding at 15° C. for 30 minutes, then further ramping up to            a temperature of 30° C. at a ramp rate of 0.5° C./minute;        -   g. the temperature was held at 30° C. for between 300 and            500 minutes; and        -   h. the vacuum was released and the lyophilizer was            backfilled with nitrogen to prevent oxidation of the            microparticle formulations before transferring into bottles            for bulk mixing and aliquoting the bulk powder for storage            at ≤−15° C.            Transfer of Bulk DAS181 Microparticles into Container and            Mixing

A section on the bottom film of each Stainless Steel tray was cleanedusing sanitizing wipes and a 3×3 cm opening was made with a scalpel. Thedry microparticles were transferred into a plastic bottle. The bottlewas capped and tumbled forty times, changing directions with eachinversion. The tumbling was to ensure uniformity of bottle content.Samples for analytical testing were taken and the bottle was recappedand sealed into plastic bags for storage at ≤−15° C.

Dry Powder Composition and Properties

The DAS181 microparticle formulation dry powder, prepared according tothe method described above as the following composition and physicalparameters:

Composition (wt/wt %):

DAS181 70.15%

Histidine free base: 6.08%

Histidine HCl: 3.92%

Trehalose: 9.25%

Citric acid: 2.54%

Sodium acetate (trace amounts from DAS181 stock solution): 0.04%

Acetic acid (trace amounts from DAS181 stock solution): 0.02%

Water: 8%

Isopropanol: trace amounts

Physical Parameters:

The DAS181 dry powder microparticles prepared according to the abovemethod have a mass median aerodynamic diameter (MMAD) of 10.4 microns, aGSD of 1.6 and an FPF of 2.1% for particle sizes<5 microns.

The suitability of the microparticles for administration by oralinhalation to treat respiratory tract infections such as influenza wastested by a Next Generation Impactor. The deposition of pharmaceuticalsin the respiratory tract can be predicted in a manner similar to that ofAndersen Cascade Impaction, by deposition of particles (microparticles)on the stages/collection plates of the cascade impactor. For the DAS181microparticles, when administered to prevent or treat viral infectionsthat initiate in the respiratory tract, such as influenza, it isdesirable to deposit the drug in the throat, trachea and bronchi (upperrespiratory airway) while avoiding deposition at the secondary andterminal bronchi and the alveoli. The results showed that only about 1%of the microparticles were deposited at the collection platescorresponding to secondary and terminal bronchi and the alveoli, with nodetectable microparticles at the collection plates corresponding toalveoli.

Packaging of the Dry Powder

The dry microparticle formulation was packaged in a primary containerclosure system that is a natural (clear), Size 3, HPMC capsule(Capsugel). The dry powder fill mass for each capsule was about 11 mg toachieve a 10 mg delivered dose. The packaged HPMC capsules were then putinto a cold form aluminum laminate blister pack as a secondary containerclosure system.

Example 3 HPMC Capsules and DAS181 Microparticles Exposed to HPMCCapsules Contain Formaldehyde

A. Qualitative Assay to Detect Aldehyde in HPMC Capsules and Dry PowderDAS181 Microparticle Formulations

The presence of aldehyde in a sample can qualitatively be detected by acolorimetric assay 4-amino-5-hydrazino-1, 2, 4-triazole-3-thiol (AHTT)(Rahn, C. H. et al., Lipids, 8(11): 612-616 (1973)). AHTT or purpald,when in basic solution, turns purple in the presence of aldehyde. In theabsence of aldehyde, the AHTT in basic solution remains slightlypinkish. The following samples were evaluated by colorimetric method:

-   -   (1) DAS181 microparticle formulations not exposed to HPMC        capsules and containing no detectable dimer    -   (2) DAS181 microparticle formulations stored in HPMC capsules        for 12 weeks at 37° C. and known to have dimer content as        determined by size exclusion HPLC (SE-HPLC or SEC)    -   (3) HPMC capsules used to package the DAS181 microparticle        formulations (#3, clear) that have never been in contact with        DAS181 microparticle formulations    -   (4) formaldehyde as a positive control.

The results showed that only samples (2), (3) and (4) turned purple whenreacted with AHTT. Thus, HPMC capsules contain aldehyde (sample (3)) andDAS181 dry powder microparticle formulations, when stored in HPMCcapsules, absorb the aldehyde that leaches from the HPMC containers(sample (2)). The DAS181 powder that never was in contact with HPMCcapsules (sample (1)) does not contain detectable amounts of aldehyde.HPMC capsule itself also contains aldehyde groups that can react withAHTT (sample (3)).

B. Formaldehyde Detection in HPMC Capsules: Quantitative Assay

The presence of extractable (leached) formaldehyde in HPMC capsules wasidentified and quantitated by GC spectroscopy. The ratio of formaldehydeto cyclohexanone internal standard was used to quantitate theextractable formaldehyde. 2, 3, 4, 5, 6-pentafluorobenzyloxamine (PFBOA)in presence of a 1% potassium hydrogen phthalate was used as aderivatizing agent.

Results from extraction of formaldehyde from HPMC capsules werereproducible in the range of 1-15 ppm formaldehyde extracted.Formaldehyde content in different capsule lots was age-dependent.Pretreatment of capsules by storing them at 40° C. and 75% RH (relativehumidity) before the analysis lowered the amount of extractableformaldehyde.

Example 4 Oligomer Formation of DAS181 in Microparticles Stored in HPMCCapsules

A. Measurement of Dimer and Higher Order Oligomer Formation

SDS-Page

The stability of DAS181 polypeptides in dry powder microparticles storedin HPMC capsules was tested by assaying for the presence of DAS181dimers and higher order oligomers formed by reacting with formaldehydefrom the HPMC capsules over time. The DAS181 dimers and higher orderoligomers can be detected qualitatively by their differential migrationon a gel relative to the monomer, as measured by SDS-PAGE. The molecularweight of DAS181 is about 45 kDa; however, due to the presence of thepositively charged amphiregulin GAG-binding domain, the DAS181 monomerband generally migrates at around 48 to 50 kDa. When dimerization ofDAS181 occurs, two dimer bands at around 100 kDa and around 120 kDa,respectively are observed. Higher order oligomers of DAS181 often stayin the well and can be detected as aggregates in the well. Thus, thestability of the DAS181 monomer in microparticle formulations can bevisualized qualitatively by SDS-PAGE.

SEC

The presence of DAS181 dimers or higher-order oligomers due to reactionof the DAS181 monomer with formaldehyde from the HPMC capsules can bequantitated by SE-HPLC or SEC (size exclusion HPLC), in which the largerdimer elutes first through the resin pores while the smaller DAS181 45kDa monomer (i.e., un-aggregated, stable form) is retained longer in thesmaller pores.

B. Stability of Microparticles in HPMC Capsules

Magnesium Sulfate as Counterion

A microparticle formulation containing magnesium sulfate as counterion,histidine as a scavenging agent and trehalose as an additionalstabilizer, was compared against other microparticle formulations whosecomposition was as follows (composition values were corrected forresidual moisture):

-   (1) Control Formulation: 96.61% DAS181, 3.10% magnesium sulfate,    0.29% calcium chloride.-   (2)+Histidine*: 80.04% DAS181, 7.72% magnesium sulfate, 0.24%    calcium chloride, 12.01% histidine-   (3)+Histidine+Trehalose: 72.71% DAS181, 5.26% magnesium sulfate,    0.22% calcium chloride, 10.91% histidine, 10.91% trehalose-   (4)+Histidine+Trehalose: 71.37% DAS181, 3.44% magnesium sulfate,    0.21%    -   +Tryptophan calcium chloride, 10.71% glycine, 3.57% tryptophan,        10.71% trehalose-   (5)+Glycine+Mannitol: 75.46% DAS181, 5.45% magnesium sulfate, 0.22%    calcium chloride, 7.55% glycine, 11.32% mannitol-   *Histidine/Histidine HCl

Formulations (1) through (5) above were placed in either a “lowformaldehyde” HPMC capsule (4 ppm formaldehyde) or a “high formaldehyde”HPMC capsule (8 ppm formaldehyde). The capsules were filled to 5 mgpowder and maintained at 37° C. for 12 weeks, at which time their dimercontent (%) was measured.

Results:

In the “low formaldehyde” HPMC capsules, the % dimer detected at 12weeks, 37° C. was as follows:

-   (1) Control Formulation: 6.9% dimer-   (2)+Histidine: 1.7% dimer-   (3)+Histidine+Trehalose: 1.7% dimer-   (4)+Histidine+Trehalose: 1.5% dimer    -   +Tryptophan-   (5)+Glycine+Mannitol: 4.8% dimer

Thus, the presence of histidine in the formulation significantlydecreased dimer formation over time and was more effective than thecombination of glycine (amine) and mannitol (sugar). While the presenceof the sugar trehalose did not appear to enhance the dimer-reducingeffect of histidine, trehalose can stabilize the protein againstmoisture-induced aggregation (see Example 6 below) by increasing thethermal stability of the formulation (due to the high glass transitiontemperature of trehalose) as well as inhibiting the reaction of freeresidual moisture with the protein.

Similar results were obtained with the “high formaldehyde” HPMCcapsules. At 12 weeks, 37° C., the % dimer detected was as follows:

-   (1) Control Formulation: 13.9% dimer-   (2)+Histidine: 1.7% dimer-   (3)+Histidine+Trehalose: 1.5% dimer-   (4)+Histidine+Trehalose: 1.5% dimer    -   +Tryptophan-   (5)+Glycine+Mannitol: 7.1% dimer

These results show that the presence of histidine in the DAS181microparticle formulations significantly reduces DAS181 dimer formationin HPMC capsules and enhances its long-term stability.

Citric Acid as Counterion

The DAS181 microparticle formulation prepared with citric acid ascounterion, histidine as a scavenging agent and trehalose as anadditional stabilizer showed a protective effect that was comparable tothat shown by the microparticle formulation of in the previous examplewith magnesium sulfate as a counterion. For example, when the sampleswere stored at 40° C. for 3 months, the percentage of DAS181 monomer inthe formulations (i. e., intact drug product) was as follows:

Formulation (magnesium sulfate counterion): 96.4%

Formulation (citric acid counterion): 96.5%

When stored at 25° C. instead of 40° C., the percentage of DAS181monomer in the formulations was as follows:

Formulation (magnesium sulfate counterion): 98.2%

Formulation (citric acid counterion): 97.9%

Example 5 Stability of DAS181 Microparticles Containing Various Amines

Microparticle formulations of DAS181 were prepared according to thegeneral method described in Example 1, except that histidine wasreplaced with various other amino acids as shown below. The dry powderformulations were stored at 37° C. in polypropylene Eppendorf tubes(unencapsulated) or in clear #3 HPMC capsules, and the amount of dimermeasured at one month, two months or three months. The results are shownbelow in Table 1 (dry powder composition values corrected for residualmoisture):

TABLE 1 Effect of Amines on DAS181 Dimer Formation in MicroparticleFormulations % Oligomer Formulation HPMC Capsules Unencapsulated (%wt/wt) Amino Acid t = 0 1 mo 2 mo 3 mo 1 mo 2 mo 3 mo 96.96% DAS181,None 0.96 11.2 7.5 10.9 4.9 5.1 6.2 2.75% sodium sulfate, 0.29% calciumchloride 97.37% DAS181, None 0.80 9.3 5.9 8.4 3.3 3.0 4.2 2.34%magnesium sulfate, 0.29% calcium chloride 84.70% DAS181, Histidine 0 1.00.9 1.2 1.3 1.1 2.2 2.04% magnesium sulfate, 0.25% calcium chloride,13.01% histidine 85.18% DAS181, Methionine 0.60 7.6 11.9 14.9 2.9 3.83.7 2.05% magnesium sulfate, 0.26% calcium chloride, 12.52% methionine85.35% DAS181, Glutamic Acid 0 4.3 3.4 3.7 1.4 1.3 2.1 2.06% magnesiumsulfate, 0.26% calcium chloride, 12.34% glutamic acid 83.19% DAS181,Arginine 0 6.0 2.3 4.5 2.2 1.3 1.9 2.0% magnesium sulfate, 0.25% calciumchloride, 14.55% arginine 91.66% DAS181, Glycine 0 4.8 2.4 3.2 3.5 2.32.9 2.21% magnesium sulfate, 0.27% calcium chloride, 5.86% glycine88.04% DAS181, Proline 0 5.0 3.2 4.1 1.9 1.7 1.7 2.12% magnesiumsulfate, 0.26% calcium chloride, 9.58% proline 80.93% DAS181, Tryptophan0.3 1.4 2.1 2.9 1.2 1.5 1.9 1.95% magnesium sulfate, 0.24% calciumchloride, 16.88% tryptophan 87.86% DAS181, Valine 0.4 7.8 12.1 16.1 2.32.8 5.2 2.12% magnesium sulfate, 0.26% calcium chloride, 9.76% valine90.36% DAS181, Alanine 0.3 6.4 8.8 12.0 1.6 1.8 2.5 2.18% magnesiumsulfate, 0.27% calcium chloride, 7.19% alanine 86.67% DAS181, Leucine0.6 7.8 12.1 14.9 3.0 3.7 4.8 2.09% magnesium sulfate, 0.26% calciumchloride, 10.98% leucine 86.67% DAS181, Isoleucine 0.5 8.1 12.1 15.3 2.73.8 4.6 2.09% magnesium sulfate, 0.26% calcium chloride, 10.98%isoleucine

Of the amines tested, histidine and tryptophan were found to be the mosteffective at preventing DAS181 oligomer formation and increasing thestability of the formulation relative to ones in which no amine ispresent. It is possible that histidine and tryptophan have a stabilizingchelating effect, in addition to the amine functionality reacting withformaldehyde.

The effect of amine concentration (% wt/wt in dry powder) on DAS181dimer formation in the microparticle formulation was measured, and theresults are shown in Table 2 (composition values corrected for residualmoisture):

TABLE 2 Effect of Amine Concentration on DAS181 Dimer Formation inMicroparticle Formulations % Oligomer Formulation HPMC CapsulesUnencapsulated (% wt/wt) Amino Acid t = 0 1 mo 2 mo 1 mo 2 mo 97.37%DAS181, None 1.1 4.8 6.9 2.6 3.4 2.34% magnesium sulfate, 0.29% calciumchloride 97.232% DAS181, 0.1% Histidine 1.2 3.5 4.4 2.4 3.1 2.3433%magnesium sulfate, 0.29% calcium chloride, 0.133% histidine 96.079%DAS181, 1.3% Histidine 1.0 2.3 3.1 2.1 2.6 2.3155% magnesium sulfate,0.2882% calcium chloride, 1.3176% histidine 91.268% DAS181, 6.26%Histidine 0.6 1.4 1.6 1.3 1.6 2.1996% magnesium sulfate, 0.2738% calciumchloride, 6.2583% histidine 84.696% DAS181, 13% Histidine 0.4 0.9 1.10.5 1.1 2.0412% magnesium sulfate, 0.2541% calcium chloride, 13.009%histidine 97.31% DAS181, 0.06% Glycine 1.1 4.9 6.6 2.5 3.2 2.35%magnesium sulfate, 0.29% calcium chloride, 0.06% glycine 96.82% DAS181,0.55% Glycine 1.1 4.3 6.0 2.6 2.1 2.33% magnesium sulfate, 0.29% calciumchloride, 0.55% glycine 94.73% DAS181, 2.7% Glycine 0.7 3.1 4.2 2.0 2.52.28% magnesium sulfate, 0.28% calcium chloride, 2.70% glycine 91.66%DAS181, 5.9% Glycine 0.5 2.3 3.8 1.7 2.1 2.21% magnesium sulfate, 0.27%calcium chloride, 5.86% glycine

The results presented in Table 2 show that for both amino acids tested,histidine and glycine, the protective effect against DAS181 dimerizationwas dependent on the wt % of the amine in the microparticle formulation.

Example 6 Stabilizing Effect of Sugars Added to DAS181 MicroparticleFormulations

The effect of various sugars on the stability of DAS181 microparticleformulations that include an amine was tested. Formulations wereprepared essentially as described in Example 1, with various sugarsbeing substituted for trehalose as indicated in Table 3 below(composition values corrected for residual moisture). Lyophilizedsamples were subjected to storage at 37° C. as bulk powder inpolypropylene Eppendorf tubes (unencapsulated) or placed in two types ofHPMC capsules: white/opaque or natural/clear. The samples were analyzedfor the presence of DAS181 dimer at the end of one month, two months orthree months using SEC.

TABLE 3 Effect of Sugars on DAS181 Dimer Formation in MicroparticleFormulations Containing Glycine % Oligomer Formulation HPMC CapsulesUnencapsulated (% wt/wt) Sugar t = 0 1 mo 2 mo 3 mo 1 mo 2 mo 3 mo77.89% DAS181, Mannitol 0 6.01 9.79 12.59 3.07 4.12 5.19 1.88% magnesium(6.87) (9.95) (12.70)* sulfate, 0.23% calcium chloride, 5% glycine, 15%mannitol 77.89% DAS181, Trehalose 0 3.75 5.82 6.79 2.19 3.13 4.05 1.88%magnesium (3.83) (5.65) (7.36)* sulfate, 0.23% calcium chloride, 5%glycine, 15% trehalose 77.89% DAS181, Sucrose 0 3.92 5.67 8.16 2.23 3.304.42 1.88% magnesium (3.98) (5.45) (7.94)* sulfate, 0.23% calciumchloride, 5% glycine, 15% sucrose 77.89% DAS181, Sorbitol 0 4.88 7.359.52 2.16 3.06 3.89 1.88% magnesium (5.01) (7.01) (9.76)* sulfate, 0.23%calcium chloride, 5% glycine, 15% sorbitol *Numbers in parentheses applyto white/opaque HPMC capsules

The results in Table 3 above indicate that trehalose and sucroseprotected DAS181 from oligomerization to a greater extent than didmannitol and sorbitol. In addition, mannitol-containing formulationswere found to contain irregular crystals, while sucrose and trehaloseform good quality microparticles.

Example 7 Effect of Antioxidants on Oligomer Formation in DAS181Microparticles

The effect of various antioxidants on the stability of DAS181microparticle formulations stored at 37° C. in HPMC capsules was testedby measuring the presence of dimer at time intervals of 0, 1.5, 3 and4.5 months. The results are shown in Table 4 below (compositionscorrected for residual moisture):

TABLE 6 Combined Effect of Amino Acids and Sugars on DAS181 Stability inMicroparticle Formulations % Oligomer at 1.5, 3.0 or 4.5 mo FormulationAmino Acid HPMC Capsules Unencapsulated (% wt/wt) and/or Sugar t = 0 1.53 4.5 1.5 3 4.5 76.86% DAS181, Histidine 0 1.0 1.6 1.6 1.4 0.9 2.8 1.85%magnesium sulfate, 0.23% calcium chloride, 21.06% histidine 65.40%DAS181, Histidine, 0 0.5 1.2 0.8 0.0 0.5 0.3 1.57% magnesium Glycine,sulfate, 0.19% Sucrose, calcium chloride, Acetate 17.92% histidine,2.55% glycine, 10.99% sucrose, 1.37% acetate 66.31% DAS181, Histidine, 00.5 1.0 0.6 0.2 0.6 0.6 1.6% magnesium Glycine, sulfate, 0.2% Sucrosecalcium chloride, 18.17% histidine, 2.59% glycine, 11.14% sucrose 68.07%DAS181, Histidine, 0 0.7 1.6 1.3 0.7 0.5 0.3 1.64% magnesium Sucrosesulfate, 0.2% calcium chloride, 18.65% histidine, 11.44% sucrose 74.62%DAS181, Histidine 0 0.8 2.1 2.0 0.7 1.3 0.6 1.8% magnesium (20.5%),sulfate, 0.22% Glycine calcium chloride, (2.9%) 20.45% histidine, 2.91%glycine 74.74% DAS181, Histidine 0 1.0 2.1 1.8 3.2 0.8 0.5 1.8%magnesium (20.5%), sulfate, 0.22% Tryptophan calcium chloride, (2.8%)20.48% histidine, 2.77% tryptophan 70.67% DAS181, Histidine 0 2.6 4.64.5 3.9 1.0 2.1 1.7% magnesium (19.4%), sulfate, 0.21% Glycine calciumchloride, (8%) 19.36% histidine, 8.06% glycine 52.66% DAS181, Histidine0 0.0 1.1 0.8 0.0 0.0 1.6 1.27% magnesium (36%), sulfate, 0.16%Tryptophan calcium chloride, (9.8%) 36.12% histidine, 9.79% tryptophan62.03% DAS181, Histidine, 0 0.6 1.3 1.0 0.2 0.4 0.1 1.49% magnesiumTrehalose sulfate, 0.18% calcium chloride, 17% histidine, 19.29%trehalose 97.37% DAS181, None 0 3.8 7.2 6.4 3.0 5.9 10.1 2.34% magnesiumsulfate, 0.29% calcium chloride

Of the antioxidants tested, ascorbic acid was found to have the mostprotective effect against DAS181 oligomer formation. Thioglycerol wasfound to enhance rather than inhibit oligomer formation. The resultsdemonstrate that antioxidants can be used to protect DAS181microparticle formulations against oligomerization in HPMC capsules.

Example 8

Effect of Chelators on Oligomer Formation in DAS181 Microparticles

The effect of various chelators on the stability of DAS181 microparticleformulations stored at 37° C. in HPMC capsules was tested by measuringthe presence of dimer at time intervals of zero, one and three months.The results are shown in Table 5 below (compositions corrected forresidual moisture):

TABLE 4 Effect of Antioxidants on DAS181 Stability in MicroparticleFormulations % Oligomer at 1.5, 3.0 or 4.5 mo Formulation HPMC CapsulesUnencapsulated (% wt/wt) Antioxidant t = 0 1.5 3 4.5 1.5 3 4.5 97.08%DAS181, Thioglycerol 0 21.1 27 20.2 23.5 24.1 28.7 2.34% magnesiumsulfate, 0.29% calcium chloride, 0.30% thioglycerol 96.88% DAS181,Ascorbic acid 0 0.4 1.4 0.8 0.1 0.8 0.5 2.33% magnesium sulfate, 0.29%calcium chloride, 0.50% ascorbic acid 97.31% DAS181, Tocopherol 0 0.63.7 3.2 0.1 3.1 5.2 2.34% magnesium (vitamin E) sulfate, 0.29% calciumchloride, 0.06% tocopherol 97.32% DAS181, Melatonin 0 2.6 4.5 5.1 4.64.4 6.0 2.34% magnesium sulfate, 0.29% calcium chloride, 0.05% melatonin96.88% DAS181, Magnesium 0 1.8 2.8 2.1 2.7 3.0 3.0 2.33% magnesiumbisulfite sulfate, 0.29% calcium chloride, 0.50% magnesium bisulfite97.37% DAS181, None 0 3.8 7.2 6.4 3.0 5.9 10.1 2.34% magnesium sulfate,0.29% calcium chloride

Of the chelators tested, TETA was the most effective at protectingDAS181 from dimer formation in the HPMC capsules, and the greater theamount of TETA in the formulation, the less the amount of oligomerformed.

Example 9 Combinations of Amino Acids and Sugars in DAS181 MicroparticleFormulations

Combinations of several amino acids with one another and/or a sugar weretested for their ability to protect DAS181 microparticles in HPMCcapsules from oligomer formation. The DAS181 microparticle formulations,prepared essentially as described in Example 1 with the ingredients setforth in Table 6 below (composition values corrected for residualmoisture), were stored at 37° C. in HPMC capsules and were tested bymeasuring the presence of DAS181 dimer (using SEC) at time intervals of0, 1.5, 3 and 4.5 months.

TABLE 5 Effect of Chelators on DAS181 Stability in MicroparticleFormulations % Oligomer Formulation Chelator - Type HPMC Capsules (10mg) Unencapsulated (% wt/wt) and wt % t = 0 1 mo 3 mo 1 mo 3 mo 84.02%DAS181, 13.7% Bicine 0 5.5 8.7 2.8 4.7 2.02% magnesium sulfate, 0.25%calcium chloride, 13.71% bicine 95.84% DAS181, 1.6% Bicine 0 6.35 10.93.1 1.9 2.31% magnesium sulfate, 0.29% calcium chloride, 1.56% bicine97.21% DAS181, 0.16% Bicine 0 5.3 9.3 2.7 5.3 2.34% magnesium sulfate,0.29% calcium chloride, 0.16% bicine 70.4% DAS181, 27.7% DTPA 0 8.3 17.62.0 1.9 1.69% magnesium sulfate, 0.21% calcium chloride, 27.69% DTPA93.78% DAS181, 3.7% DTPA 0 4.75 8.4 2.4 4.0 2.26% magnesium sulfate,0.28% calcium chloride, 3.69% DTPA 97% DAS181, 0.38% DTPA 0 5.0 8.1 2.24.9 2.33% magnesium sulfate, 0.29% calcium chloride, 0.38% DTPA 85.23%DAS181, 12.5% TETA 0 0.8 1.8 0.7 1.7 2.05% magnesium sulfate, 0.25%calcium chloride, 12.46% TETA 96% DAS181, 1.4% TETA 0 1.3 2.9 0.9 2.72.31% magnesium sulfate, 0.29% calcium chloride, 1.4% TETA 97.23%DAS181, 0.14% TETA 0 2.5 4.4 1.9 5.3 2.34% magnesium sulfate, 0.29%calcium chloride, 0.14% TETA 97.37% DAS181, None 0 5.5 8.7 2.8 4.7 2.34%magnesium sulfate, 0.29% calcium chloride

While histidine alone clearly had a protective effect against DAS181oligomerization in HPMC capsules, some combinations of amino acids andsugars provided better protection in the microparticle formulations,e.g., Histidine/Glycine/Sucrose, Histidine/Trehalose andHistidine/Tryptophan especially at the higher concentration oftryptophan (9.8%).

DAS 181 (without amino terminal Met) (SEQ ID NO: 1)GDHPQATPAPAPDASTELPASMSQAQHLAANTATDNYRIPAITTAPNGDLLISYDERPKDNGNGGSDAPNPNHIVQRRSTDGGKTWSAPTYIHQGTETGKKVGYSDPSYVVDHQTGTIFNFHVKSYDQGWGGSRGGTDPENRGIIQAEVSTSTDNGWTWTHRTITADITKDKPWTARFAASGQGIQIQHGPHAGRLVQQYTIRTAGGAVQAVSVYSDDHGKTWQAGTPIGTGMDENKVVELSDGSLMLNSRASDGSGFRKVAHSTDGGQTWSEPVSDKNLPDSVDNAQIIRAFPNAAPDDPRAKVLLLSHSPNPRPWSRDRGTISMSCDDGASWTTSKVFHEPFVGYTTIAVQSDGSIGLLSEDAHNGADYGGIWYRNFTMNWLGEQCGQKPAKRKKKGGKNGKNRRNRKKKNP DAS 181 (without amino terminal Met)(SEQ ID NO: 2) MGDHPQATPAPAPDASTELPASMSQAQHLAANTATDNYRIPAITTAPNGDLLISYDERPKDNGNGGSDAPNPNHIVQRRSTDGGKTWSAPTYIHQGTETGKKVGYSDPSYVVDHQTGTIFNFHVKSYDQGWGGSRGGTDPENRGIIQAEVSTSTDNGWTWTHRTITADITKDKPWTARFAASGQGIQIQHGPHAGRLVQQYTIRTAGGAVQAVSVYSDDHGKTWQAGTPIGTGMDENKVVELSDGSLMLNSRASDGSGFRKVAHSTDGGQTWSEPVSDKNLPDSVDNAQIIRAFPNAAPDDPRAKVLLLSHSPNPRPWSRDRGTISMSCDDGASWTTSKVFHEPFVGYTTIAVQSDGSIGLLSEDAHNGADYGGIWYRNFTMNWLGEQCGQKPAKRKKKGGKNGKNRRNRKKKNP

1.-78. (canceled)
 79. A method for preparing a pharmaceuticalcomposition comprising DAS181, the method comprising: a) providingmicroparticles comprising DAS181 having the amino acid sequence of SEQID NO:1 or SEQ ID NO:2, wherein the microparticles are about 60-70%wt/wt DAS181, 7-15% wt/wt histidine, 7-11% wt/wt trehalose, 4-8% wt/wtmagnesium sulfate, and 8-12% wt/wt water, wherein the microparticleshave a Mass Median Aerodynamic Diameter (MMAD) of 3-8 microns; and b)combining the microparticles with a pharmaceutically acceptable aqueoussolution.
 80. The method of claim 79, wherein the microparticles are 62%to 68% (wt/wt) DAS181.
 81. The method of claim 79, wherein themicroparticles have a Geometric Standard Deviation (GSD) of 1.5-1.7,1.3-1.6, or 1.4-1.6.
 82. The method of claim 79, wherein the weightpercent of microparticles having a fine particle fractionation (FPF)below 5 microns is less than 10%.
 83. A method for preparing apharmaceutical composition comprising DAS181, the method comprising: a)providing microparticles comprising DAS181 having the amino acidsequence of SEQ ID NO:1 or SEQ ID NO:2, wherein the microparticles areabout 69-74% wt/wt DAS181, 9-17% wt/wt histidine, 8-12% wt/wt trehalose,and 4-8% wt/wt magnesium sulfate; and b) combining the microparticleswith a pharmaceutically acceptable aqueous solution.
 84. The method ofclaim 83, wherein the microparticles have a Geometric Standard Deviation(GSD) of 1.5-1.7, 1.3-1.6, or 1.4-1.6.
 85. The method of claim 83,wherein the weight percent of microparticles having a fine particlefractionation (FPF) below 5 microns is less than 10%.
 86. A method forpreparing a pharmaceutical composition comprising DAS181, the methodcomprising: a) providing microparticles comprising DAS181 having theamino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, wherein themicroparticles are about 80-90% wt/wt DAS181, 1.5-3.5% wt/wt sodiumsulfate, and 8-12% water; and b) combining the microparticles with apharmaceutically acceptable aqueous solution.
 87. The method of claim86, wherein the microparticles have a Geometric Standard Deviation (GSD)of 1.2-1.8, 1.3-1.7 or 1.4-1.6.
 88. The method of claim 86, wherein theweight percent of microparticles having a fine particle fractionation(FPF) below 5 microns is less than 10%.
 89. The method of claim 79,wherein the microparticles comprise 3-6% wt/wt histidine free base and5-9% wt/wt histidine hydrochloride.
 90. The method of claim 83, whereinthe microparticles comprise 3-8% wt/wt histidine free base and 5-9%wt/wt histidine hydrochloride.
 91. A method for preparing apharmaceutical composition comprising DAS181, the method comprising: a)providing microparticles comprising DAS181 having the amino acidsequence of SEQ ID NO:1 or SEQ ID NO:2, wherein the microparticles areabout 60-70% wt/wt DAS181, 8-15% wt/wt histidine, 7-11% wt/wt trehalose,4-8% wt/wt magnesium sulfate, and 8-12% water; and b) combining themicroparticles with a pharmaceutically acceptable aqueous solution. 92.A method for preparing a pharmaceutical composition comprising DAS181,the method comprising: (a) providing microparticles comprising DAS181having the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, whereinthe microparticles are about 69-74% wt/wt DAS181, 7-17% wt/wt histidine,8-12% wt/wt trehalose, and 4-8% wt/wt magnesium sulfate; and b)combining the microparticles with a pharmaceutically acceptable aqueoussolution.